CN109055566A - The detection method and application thereof of cow group HH type genetic defect gene - Google Patents
The detection method and application thereof of cow group HH type genetic defect gene Download PDFInfo
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- CN109055566A CN109055566A CN201810762088.9A CN201810762088A CN109055566A CN 109055566 A CN109055566 A CN 109055566A CN 201810762088 A CN201810762088 A CN 201810762088A CN 109055566 A CN109055566 A CN 109055566A
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Abstract
The present invention relates to a kind of detection methods of cow group HH type genetic defect gene, include the following steps: S1: the blood sample for obtaining cow group to be detected extracts DNA sample using DNA extraction kit from blood sample；S2: using designed KASP primer, carries out PCR amplification to DNA sample；Wherein, KASP primer includes three, respectively upstream normal gene primer, upstream mutated gene primer and downstream primer, also, in upstream normal gene primer and upstream mutated gene primer, and 5,End is respectively designed with HEX fluorescence labels sequence and FAM fluorescence labels sequence；S3: according to the fluorescence intensity of the DNA sample after amplification, judge the genotype of the HH type gene in corresponding cow group.The present invention establishes the genetic defect gene for detecting using KASP classifying method and influencing cow reproduction power for the first time, and this method is quick, efficient, economical, and high-throughput detection can be carried out to sample.
Description
Technical field
The present invention relates to molecular biology and Biotechnology in Genetic Breeding fields, and in particular to a kind of cow group HH type heredity is scarce
Fall into the detection method and application thereof of gene.
Background technique
In recent years, in order to improve the production performance of milk cow, China largely introduces inhereditary material from foreign countries, promotes domestic milk
The genetic improvement of cows body.However, also resulting in some genetic defectes during introducing inhereditary material in China milk cows
The propagation of body.On October 28th, 2016, animal husbandry department of the Ministry of Agriculture have issued " The Ministry of Agriculture of the People's Republic of China, MOA's bulletin the 2460th
Number ", regulation should identify or in sales contract clause from overseas introduced variety ox and frozen semen and embryo on pedigree in bulletin
In write kind of ox and frozen semen and embryo exactly and do not carry spinal curvature syndrome (CVM), leukocyte adhesion deficiency syndrome
(BLAD), the central genetics dcc gene such as short spinal syndromes (BY).Therefore kind of an ox dcc gene detection platform is established, to guarantee
The quality safety of kind ox and inhereditary material, the economic benefit for improving dairy farmer all have significance.
2011, VanRaden et al. utilize North America cows 50K Illumina SNPs genotype data, by software with
And pedigree analysis, and by the association analysis with specific reproductive trait (SCR, pregnancy rate, still birth rate), it is sent out in Holstein cow group
Three kinds of recessive inheritance defect haplotypes (HH1, HH2 and HH3) for leading to embryonic death are showed, HH1 type genetic defect is APAF1 base
Caused by mutation, HH3 type genetic defect is that SMC2 gene mutation causes, and when its homozygosis, will lead to early abortion or stillborn foetus, and
Heterozygote carrier will not show any symptom, therefore, it is necessary to heterozygote carrier is accurately detected and screened,
Offspring is avoided to occur miscarrying or the phenomenon that stillborn foetus.
KASP is the abbreviation of competitive ApoE gene, can be in extensive genome DNA sample, to SNPs
Accurately diallele is carried out with the InDels (Insertions and Deletions, insertion and missing) on specific site
Judgement is to carry out parting to SNP based on the special matching of prime end base.The side of detection milk cow genetic defect gene at present
Method mainly has PCR-PIRA, PCR-SSCP, PCR-RFLP and pyrosequencing, does not have using KASP technology for detecting milk cow
The report of HH type genetic defect gene.
Summary of the invention
It is not deposited for solution is of the existing technology, the present invention provides a kind of inspections of cow group HH type genetic defect gene
Survey method, includes the following steps:
Step S1: the blood sample for obtaining cow group to be detected is mentioned from blood sample using DNA extraction kit
Take DNA sample；
Step S2: using designed KASP primer, carries out PCR amplification to DNA sample；
Wherein, KASP primer includes three, respectively upstream normal gene primer, upstream mutated gene primer and downstream
Primer, also, in upstream normal gene primer and upstream mutated gene primer, 5 ' ends are respectively designed with HEX fluorescence labels sequence
Column and FAM fluorescence labels sequence；
Step S3: according to the fluorescence intensity of the DNA sample after amplification, judge that the HH type heredity in corresponding cow group lacks
Fall into the genotype of corresponding gene.
Wherein, HH type genetic defect gene detected includes HH1 and HH3 type genetic defect gene, wherein HH1 type is lost
It passes dcc gene to be caused by APAF1 gene mutation, HH3 type genetic defect gene is caused by SMC2 gene mutation.
Wherein, when detecting to HH1 type genetic defect gene, designed primer is as follows:
Upstream normal gene primer: 5 '-GAAGGTCGGAGTCAACGGATTGAAACTTCAGAGGTTTAT CGGC-3 '；
Upstream mutated gene primer: 5 '-GAAGGTGACCAAGTTCATGCTGAAACTTCAGAGGTTTAT CGGT-3 '；
Downstream primer: 5 '-GGTAAAGCATTCCGTTATCGAC-3 '.
Wherein, when detecting to HH3 type genetic defect gene, designed primer is as follows:
Upstream normal gene primer: 5 '-GAAGGTCGGAGTCAACGGATTTGGACATATGCTACGTACT CATTT-
3'；
Upstream mutated gene primer: 5 '-GAAGGTGACCAAGTTCATGCTTGGACATATGCTACGTACT CATTC-
3'；
Downstream primer: 5 '-AAATCCTAGAACTATTCTGAGGCTC-3 '.
Wherein, in the step S2, PCR amplification system includes: the mix primer of 10-15 parts of volume contents, 45-50 parts of bodies
The DNA sample of product content and the KASP mixed liquor of 45-50 parts of volume contents, blank control replace DNA sample with double distilled water.
Wherein, in the step S2, PCR amplification includes the following steps:
Step S21: it is denaturalized 18-22s at 90-95 degrees Celsius of lower hot activation 14-16min, 90-95 degrees Celsius, 61-55 takes the photograph
It anneals under family name's degree and extends 55-65s, carry out 10 circulations；
Step S22: being denaturalized 18-22s under 90-95 degrees Celsius, anneals under 54-56 degrees Celsius and extends 55-65s, carries out
26 circulations；
Step S23: being denaturalized 18-22s under 90-95 degrees Celsius, anneals under 54-59 degrees Celsius and extends 55-65s, carries out 3
A circulation.
Wherein, in the step S2, PCR amplification includes the following steps:
Step S21: being denaturalized 20s at 94 degrees Celsius of lower hot activation 15min, 94 degrees Celsius, under 61-55 degrees Celsius annealing and
Extend 60s, carries out 10 circulations；
Step S22: being denaturalized 20s under 94 degrees Celsius, anneals under 55 degrees Celsius and extends 60s, carries out 26 circulations；
Step S23: being denaturalized 20s under 94 degrees Celsius, anneals under 57 degrees Celsius and extends 60s, carries out 3 circulations.
Invention additionally provides a kind of use of the detection method of cow group HH type genetic defect gene as described above
On the way, the detection method of the cow group HH type genetic defect gene is used for the genetic defect genetic test and science of holstein cows
Seed selection and selective pairing.
The detection method and purposes of cow group HH type genetic defect gene provided by the invention, establish use for the first time
The detection of KASP genotyping detection method influences the genetic defect gene of cow reproduction power, and this method is quick, efficient, economical, can be to sample
The detection of this progress high throughput.
Detailed description of the invention
The KASP testing result figure of Fig. 1: HH1 genetic defect gene.
Specific embodiment
In order to have further understanding to technical solution of the present invention and beneficial effect, it is described in detail with reference to the accompanying drawing
Technical solution of the present invention and its beneficial effect of generation.
The present invention is directed to the mutated individuals of APAF1 gene and SMC2 gene in accurate, quick detection holstein cows, thus
Reference is provided for scientific seed selection and selective pairing.
1. test specimen
The blood sample of 158 holstein cows.
2. extracting genome DNA
Using commercially available DNA extraction kit, according to kit specification, extract in every cow head blood sample
DNA sample.
The foundation of 3.KASP detection method
3.1 design of primers
The cow genome group sequence (UMD 3.1) of Ensembl database is retrieved, the partial sequence of APAF1 gene is obtained
(BTA5:63150200-63150600) and the partial sequence of SMC2 gene (BTA8:95410307-95410707), with this
PCR primer design is carried out for template.
The partial sequence of APAF1 gene is as follows:
GATGGTATAG ACTGTGAGAA TTTCCAGGAG TTTTTATCTT TAAATGGACA TCTTCTTGGA
GACAGCCATT TCCTAATATT GTGCAACTGG GCCTCTGTGA ACTGGAAACT TCAGAGGTTT ATCGG[C/T]
AAGCT AAGCTGCAGG CCAAGCAGGA GGTCGATAAC GGAATGCTTT ACCTGGAGTG GGT。
Wherein, to design corresponding part when upstream primer and downstream primer, upstream primer is corresponding for underscore mark part
The last one partial gene point is the point that gene mutation occurs, corresponding:
Upstream primer F1:5 '-AAACTTCAGAGGTTTATCGGC-3 '；
Upstream primer F2:5 '-AAACTTCAGAGGTTTATCGGT-3 '；
Downstream primer: 5 '-GGTAAAGCATTCCGTTATCGAC-3 '.
Table 1 is shown for primer designed by APAF1 gene, for SNP site, upstream primer APAF1-F1 and
APAF1-F2 is respectively the primer designed according to upstream normal gene and mutated gene, and the 3 ' of two primers are held dashes forward for allele
Become base (C or T of italicized item), APAF1-F1 and APAF1-F2 is added corresponding universal joint at 5 ' ends simultaneously by the present invention
Sequence (fluorescence labels sequence) is as follows to facilitate the identification of later period different genotype:
The KASP primer sequence information of 1 APAF1 gene of table
The partial sequence of SMC2 gene is as follows:
TCTACATCCT GGATGAGGTC GATGCAGCCC TGGATCTTTC TCATACTCAG
AATATTGGACATATGCTACG TACTCATT[T/C]C ACACATTCTC AGGTAAGAAC CAAAAAGAGC CTCAGAATAGTTCTAGGATT TGTTTTTCTA AAACTATTCT TTAGTAATGG TCAGTATATA
TAAGGAATTTAGGATTGAAG AATACCAAGG TGTTGTATAT CTCTTTAAAT ATATAATTCT ATTCATATGG。
Wherein, to design corresponding part when upstream primer and downstream primer, upstream primer is corresponding for underscore mark part
The last one partial gene point is the point that gene mutation occurs, corresponding:
Upstream primer F1:5 '-TTGGACATATGCTACGTACTCATTT-3 '；
Upstream primer F2:5 '-TTGGACATATGCTACGTACTCATTC-3 '；
Downstream primer: 5 '-AAATCCTAGAACTATTCTGAGGCTC-3 '.
Table 2 is shown for primer designed by SMC2 gene, for SNP site, upstream primer SMC2-F1 and SMC2-
F2 is respectively the primer designed according to upstream normal gene and mutated gene, and the 3 ' of two primers are held as allelic mutation base
(C or T of italicized item), the present invention are (glimmering plus corresponding universal linker sequence at 5 ' ends by SMC2-F1 and SMC2-F2 simultaneously
Optical label sequence), as follows to facilitate the identification of later period different genotype:
The KASP primer sequence information of 2 SMC2 gene of table
3.2 PCR programs
PCR system is 5 μ L, including 2.5 μ L DNA, 2.5 μ L KASP Master Mix (KASP mixed liquor) (LGC
Genomics, Hoddeston, UK), 0.07 μ L mix primer.To avoid the erroneous judgement to test result, blank control is set,
DNA profiling ddH2O circulation；It anneals when 94 DEG C of time variation 20s, 57 DEG C and extends 60s, carries out 3 circulations.Instead of.PCR amplification
Condition temperature be 94 DEG C when hot activation 15min, 94 DEG C of time variation 20s, 61-55 DEG C when anneal and extend 60s, carry out 10
Circulation；It anneals when 94 DEG C of time variation 20s, 55 DEG C and extends 60s, carries out 26
3.3 data scanning
After PCR amplification, for amplification, data scanning is carried out, specifically can refer to " ABI7900HT Fast
RealTime PCR system experimental implementation regulation ", Fig. 1 is the HH1 genetic defect gene that holstein cows are directed in the present invention
KASP testing result figure, as shown in Figure 1, the upper left corner display portion milk cow individual in DNA sample amplification after fluorescence intensity, this
The genetic entities of part corresponding homozygous unmutated (CC), since amplified band has HEX fluorescence labels sequence, the DNA sample after amplification
Blue, the fluorescence intensity in lower right corner display portion milk cow individual after DNA sample amplification, this part pair are presented after this fluorescent scanning
The genetic entities for answering heterozygous mutant (CT), since amplified band had both had HEX fluorescence labels sequence or had FAM fluorescence labels sequence
It arranges, the secondary colour of two kinds of fluorescence labels corresponding colors is presented after the DNA sample fluorescent scanning after amplification, i.e., red and blue mixes
Green afterwards.
4. genotype call results
4.1 group's Genome ratios calculate
Heterozygous genotypes frequency (%)=heterozygous individual number/measurement number of individuals × 100%
Deleterious gene frequency=heterozygous genotypes frequency/2
4.2 genotype call results
The present invention is total to 158 holstein cows of random detection, for APAF1 gene, detects 20 carrier altogether
Body, HH1 carrying rate are 12.66%, and deleterious gene frequency is 6.33%.For SMC2 gene, 6 carrier are detected altogether
Body, HH3 carrying rate are 3.8%, and deleterious gene frequency is 1.9%.
It is so-called " FAM " in the present invention, refer to hydroxyl fluorescein；So-called " HEX " refers to chlordene fluorescein amino phosphorus
Acid esters.
Beneficial effects of the present invention are as follows:
1, using the carrier of HH1 and HH3 type genetic defect gene in KASP detection cow, recall rate reaches 100%.
2, it is detected using the method for KASP, compares more existing detection method, more rapidly, efficiently and accurately.
3, detection method of the invention provides foundation for scientific seed selection and selective pairing, has to the economic loss for reducing cattle breeding
It is significant.
Although the present invention is illustrated using above-mentioned preferred embodiment, the protection model that however, it is not to limit the invention
It encloses, anyone skilled in the art are not departing within the spirit and scope of the present invention, and opposite above-described embodiment carries out various changes
It is dynamic still to belong to the range that the present invention is protected with modification, therefore protection scope of the present invention subjects to the definition of the claims.
Claims (8)
1. a kind of detection method of cow group HH type genetic defect gene, which comprises the steps of:
Step S1: the blood sample for obtaining cow group to be detected extracts DNA using DNA extraction kit from blood sample
Sample；
Step S2: using designed KASP primer, carries out PCR amplification to DNA sample；
Wherein, KASP primer includes three, and respectively upstream normal gene primer, upstream mutated gene primer and downstream is drawn
Object, also, in upstream normal gene primer and upstream mutated gene primer, 5,End is respectively designed with HEX fluorescence labels sequence
And FAM fluorescence labels sequence；
Step S3: according to the fluorescence intensity of the DNA sample after amplification, judge the HH type genetic defect pair in corresponding cow group
The genotype for the gene answered.
2. the detection method of cow group HH type genetic defect gene as described in claim 1, it is characterised in that: detected
HH type genetic defect gene includes HH1 and HH3 type genetic defect gene, wherein HH1 type genetic defect gene is by APAF1 gene
Mutation causes, and HH3 type genetic defect gene is caused by SMC2 gene mutation.
3. the detection method of cow group HH type genetic defect gene as claimed in claim 2, it is characterised in that: to HH1
When type genetic defect gene is detected, designed primer is as follows:
Upstream normal gene primer: 5 '-GAAGGTCGGAGTCAACGGATTGAAACTTCAGAGGTTTAT
CGGC-3'；
Upstream mutated gene primer: 5 '-GAAGGTGACCAAGTTCATGCTGAAACTTCAGAGGTTTAT
CGGT-3'；
Downstream primer: 5 '-GGTAAAGCATTCCGTTATCGAC-3 '.
4. the detection method of cow group HH type genetic defect gene as claimed in claim 2, it is characterised in that: to HH3
When type genetic defect gene is detected, designed primer is as follows:
Upstream normal gene primer: 5 '-GAAGGTCGGAGTCAACGGATTTGGACATATGCTACGTACT
CATTT-3'；
Upstream mutated gene primer: 5 '-GAAGGTGACCAAGTTCATGCTTGGACATATGCTACGTACT
CATTC-3'；
Downstream primer: 5 '-AAATCCTAGAACTATTCTGAGGCTC-3 '.
5. the detection method of cow group HH type genetic defect gene as described in claim 1, it is characterised in that: the step
In S2, PCR amplification system includes: the mix primer of 10-15 parts of volume contents, the DNA sample of 45-50 parts of volume contents and
The KASP mixed liquor of 45-50 parts of volume contents, blank control replace DNA sample with double distilled water.
6. the detection method of cow group HH type genetic defect gene according to any one of claims 1 to 5, feature exist
In: in the step S2, PCR amplification includes the following steps:
Step S21: being denaturalized 18-22s at 90-95 degrees Celsius of lower hot activation 14-16min, 90-95 degrees Celsius, and 61-55 degrees Celsius
Lower annealing and extension 55-65s, carry out 10 circulations；
Step S22: being denaturalized 18-22s under 90-95 degrees Celsius, anneals under 54-56 degrees Celsius and extends 55-65s, carries out 26
Circulation；
Step S23: being denaturalized 18-22s under 90-95 degrees Celsius, anneals under 54-59 degrees Celsius and extends 55-65s, carries out 3 and follow
Ring.
7. the detection method of cow group HH type genetic defect gene as claimed in claim 6, it is characterised in that: the step
In S2, PCR amplification includes the following steps:
Step S21: it is denaturalized 20s at 94 degrees Celsius of lower hot activation 15min, 94 degrees Celsius, anneals and extends under 61-55 degrees Celsius
60s carries out 10 circulations；
Step S22: being denaturalized 20s under 94 degrees Celsius, anneals under 55 degrees Celsius and extends 60s, carries out 26 circulations；
Step S23: being denaturalized 20s under 94 degrees Celsius, anneals under 57 degrees Celsius and extends 60s, carries out 3 circulations.
8. the purposes of the detection method such as cow group HH type genetic defect gene of any of claims 1-7,
Be characterized in that: the detection method of the cow group HH type genetic defect gene is used for the genetic defect genetic test of holstein cows
And scientific seed selection and selective pairing.
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Cited By (1)
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| CN114032314A (en) * | 2021-12-14 | 2022-02-11 | 广西壮族自治区水牛研究所 | Method for detecting river-type buffalo beta-casein genotype based on SNP technology |
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| CN105087571A (en) * | 2015-09-09 | 2015-11-25 | 中国农业大学 | Molecular detection method for screening complex vertebral malformation carried Holstein cows |
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| CN114032314A (en) * | 2021-12-14 | 2022-02-11 | 广西壮族自治区水牛研究所 | Method for detecting river-type buffalo beta-casein genotype based on SNP technology |
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