CN113774153A - Molecular marker for identifying Guizhou black goat and le-to-black goat, detection method and application - Google Patents
Molecular marker for identifying Guizhou black goat and le-to-black goat, detection method and application Download PDFInfo
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- CN113774153A CN113774153A CN202111248668.4A CN202111248668A CN113774153A CN 113774153 A CN113774153 A CN 113774153A CN 202111248668 A CN202111248668 A CN 202111248668A CN 113774153 A CN113774153 A CN 113774153A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a molecular marker for identifying Guizhou black goats and Happy to black goats, a detection method and application, and belongs to the technical field of molecular detection. The invention discloses a molecular marker, the nucleotide sequence of which is shown as SEQ ID NO: 1 and SEQ ID NO: 1 is G at position 97 and C at position 232, the goat is Guizhou black goat; t at position 97, and a le-to-black goat at position 232. The invention discovers polymorphic sites capable of being used for identifying two varieties by analyzing the gene difference of INHBB genes in Guizhou black goats and le-black goats, and realizes accurate distinction and identification of the Guizhou black goats and le-black goats through designed primer pairs based on the polymorphic sites, which has important significance for pure breeding and breeding local variety goats by using molecular means.
Description
Technical Field
The invention relates to the technical field of molecular detection, in particular to a molecular marker for identifying Guizhou black goats and le-to-black goats, a detection method and application.
Background
With the development of the mutton sheep industry, the demand for pure breeding of local varieties is increasing, and Guizhou black goats and le-black goats belong to goat varieties unique in southwest regions and belong to local goat variety resources in national livestock and poultry variety resources. The Guizhou black goat and the Leyou black goat have the characteristics of similar body shapes and appearances, but some genes with special functions of local goat germplasm resources need to be screened and identified in the pure breeding and breeding processes of the goats, and particularly, functional genes related to the goat lambing rate are subjected to accurate breeding. The breeding of the sheep comprises two aspects of pure breeding and hybridization, wherein the pure breeding and the hybridization need pure breeding sheep, and good breeding sheep can breed offspring with excellent quality. However, the conventional breeding methods are mostly adopted in the current breeding and breeding processes, such as: the natural breeding is combined with the hybridization mode to carry out pure breeding and variety breeding, the breeding and breeding mode needs long period and is not beneficial to the retention of dominant genes, and breeding sheep and offspring with excellent characters are difficult to accurately select. With the development of molecular technology, the molecular technology has been applied to different fields, but the molecular identification technology for Guizhou black goats and Happy to black goats is relatively deficient. Therefore, a molecular biological method capable of accurately identifying Guizhou black goats and Happy to black goats is needed to solve the existing problems.
Disclosure of Invention
The invention aims to provide a molecular marker, a detection method and application for identifying Guizhou black goats and le-to-black goats, so as to solve the problems in the prior art, and the designed primer pair can realize accurate identification and discrimination of the Guizhou black goats and le-to-black goats, and has important significance for pure breeding and breeding of goats.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a molecular marker for identifying Guizhou black goats and Happy to black goats, which has a nucleotide sequence shown as SEQ ID NO: 1 and SEQ ID NO: 1 is G at position 97 and C at position 232, the goat is Guizhou black goat; t at position 97, and a le-to-black goat at position 232.
The invention also provides a primer pair for amplifying the molecular marker, which comprises:
an upstream primer: 5'-ACGAAGGCAACCAGAACCTG-3', respectively;
a downstream primer: 5'-CGCAATGATCCAGTCGTTCC-3' are provided.
The invention also provides a kit for identifying Guizhou black goats and le-black goats, which comprises the primer pair.
The invention also provides a molecular detection method for identifying Guizhou black goats and Happy to black goats, which comprises the following steps: taking the genomic DNA of the black goat to be identified as a template, performing PCR amplification by using the primer pair, and determining the variety of the black goat to be identified according to the obtained amplification product by the following method:
if the sequence of the amplification product of the black goat to be identified is shown as SEQ ID NO: 1, and if the 97 th position is G and the 232 th position is C, the goat is judged to be Guizhou black goat; if the sequence of the amplification product of the black goat to be identified is shown as SEQ ID NO: and 1, if the 97 th position is T and the 232 th position is T, the goat is judged to be le to black.
Preferably, the 25. mu.L PCR amplification reaction system comprises: PCR mixture 12. mu.L, template 2. mu.L of upstream and downstream primers 1. mu. L, cDNA, respectively, and double distilled water 9. mu.L.
Preferably, the PCR amplification reaction procedure is: pre-denaturation at 95 ℃ for 5 min; at 94 ℃ for 40s, at 53 ℃ for 40s, at 72 ℃ for 50s, for 35 cycles; extension at 72 ℃ for 10 min.
The invention also provides application of the molecular marker or the primer pair or the kit or the molecular detection method in identifying Guizhou black goats and le-black goats.
The invention also provides application of the molecular marker or the primer pair or the kit or the molecular detection method in pure breeding and selective breeding of Guizhou black goats and/or Happy to black goats.
The invention discloses the following technical effects:
the invention utilizes the difference of INHBB gene sequences to find the polymorphic sites capable of accurately identifying Guizhou black goat and le to black goat, and the primers are designed based on the polymorphic sites, so that the fast and accurate identification of two different varieties of Guizhou black goat and le to black goat can be realized, and the molecular identification means is provided for accurately screening the specific functional genes in the local goat germplasm resources in the pure breeding and breeding process of local variety goats, and the basis is provided for the reservation of local goat variety dominant genes. In addition, the design of a kit containing the primer designed based on the polymorphic site facilitates the operation and implementation of a basic layer, and can generate considerable economic benefit and use value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a nucleotide sequence comparison diagram of the INHBB gene of Guizhou black goat and le-Hei goat.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
Establishment of molecular detection method for identifying Guizhou black goat and le-black goat
1. Primer design
The applicant compares the nucleotide sequences of INHBB genes (Guizhou black goat: NCBI Accession No: PRJNA613301, le zhi black goat: NCBI Accession No: JN620338.1) of Guizhou black goat and le zhi black goat, finds that the two goat varieties have variation at the 97 th site and the 232 th site of the nucleotide sequences (see figure 1), and designs a primer to amplify the candidate site based on the difference.
Upstream primer (SEQ ID NO: 2): 5'-ACGAAGGCAACCAGAACCTG-3', respectively;
downstream primer (SEQ ID NO: 3): 5'-CGCAATGATCCAGTCGTTCC-3' are provided.
2. PCR amplification
Selecting Guizhou black goat and Happy goat with similar body and appearance, cutting a small piece of ear tissue by using an ear clamp, extracting total mRNA by a trozol method, carrying out reverse transcription to obtain cDNA, and carrying out amplification by using designed primers.
A25. mu.L PCR amplification reaction system included: PCR mixture 12. mu.L, template 2. mu.L of upstream and downstream primers 1. mu. L, cDNA, respectively, and double distilled water 9. mu.L.
The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; at 94 ℃ for 40s, at 53 ℃ for 40s, at 72 ℃ for 50s, for 35 cycles; extension at 72 ℃ for 10 min.
3. Electrophoretic detection
The PCR product was detected by 1.5% agarose gel electrophoresis, and a specific band appeared at 715bp, whose fragment size was consistent with the expected result. Sending the detected PCR product to Hunan Optimalaceae biology Limited company to perform Sanger direct sequencing by using an upstream primer, comparing sequencing sequences, finding that variation exists at the 97 th site and the 232 th site of INHBB gene, and obtaining Guizhou black goat only when the 97 th site nucleotide is G and the 232 th site nucleotide is C; the nucleotide at position 97 is T, and when the nucleotide at position 232 is T, the goat is le to black.
Example 2
50 Guizhou black goats and Lezao black goats were selected respectively, and both varieties were from animal husbandry and veterinary research institute of Guizhou province.
The results of identifying the gene distributions of the above two varieties by the molecular assay method of the present invention are shown in Table 1.
TABLE 1 identification results
As can be seen from Table 1, the black goat in Guizhou is obtained when GA is at position 97 and CC/CT is at position 232, or GG is at position 97 and CC/CT is at position 232; and when the 97 th position is TC and the 232 th position is TC/TT, or the 97 th position is TT and the 232 th position is TC/TT, the goat is le-black goat.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> animal husbandry veterinary institute of Guizhou province
<120> molecular marker for identifying Guizhou black goat and le-black goat, detection method and application
<160> 3
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caacgaaggc aaccagaacc tgtttgtagt gcaggccagc ctgtggctct acctgaagct 60
gctgccctac gtcctggaga agggcggccg gcggaaggtg cgggtaaagg tgtacttcca 120
ggagcagggc cctggcgacc gctgggccgc ggtggagaag cgcgtggacc tcaagcgcag 180
cggctggcac accttccccc tcaccgaacc catccaggcc ctgttctcac gcggcgagcg 240
gcggctcagc ctggacgtgc agtgcgacgg ctgccgggag ctggccgtgg tgcccgtgtt 300
cgtggacccg ggcgaggagt cgcaccggcc cttcgtggtg gtgcaggcgc ggctgggcga 360
cagcaggcac cgcatccgca agcggggcct ggagtgcgac gggaggacca acctgtgctg 420
ccggcagcag ttcttcatcg acttccgcct catcggctgg aacgactgga tcattgcgcc 480
caccggctac tatgggaact actgcgaggg cagctgccct gcctacctgg cgggggtgcc 540
gggctcggcc tcctccttcc acacggccgt ggtaaaccag taccgcatgc gggggctgaa 600
cccgggcacc gtgaactcct gctgtatccc caccaagctg agcaccatgt ccatgctcta 660
cttcgacgac gagtacaaca tcgtcaagcg ggacgtgccc aatatgatcg tggagg 716
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<213> Artificial Sequence (Artificial Sequence)
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acgaaggcaa ccagaacctg 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cgcaatgatc cagtcgttcc 20
Claims (8)
1. A molecular marker for identifying Guizhou black goats and Happy to black goats is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1 and SEQ ID NO: 1 is G at position 97 and C at position 232, the goat is Guizhou black goat; t at position 97, and a le-to-black goat at position 232.
2. A primer pair for amplifying the molecular marker of claim 1, comprising:
an upstream primer: 5'-ACGAAGGCAACCAGAACCTG-3', respectively;
a downstream primer: 5'-CGCAATGATCCAGTCGTTCC-3' are provided.
3. A kit for identifying a black goat of Guizhou and a black goat of Happy to Hakka, comprising the primer set of claim 2.
4. A molecular detection method for identifying Guizhou black goats and le-black goats is characterized by comprising the following steps: taking the genomic DNA of the black goat to be identified as a template, carrying out PCR amplification by using the primer pair of claim 2, and determining the variety of the black goat to be identified according to the following method by using the obtained amplification product:
if the sequence of the amplification product of the black goat to be identified is shown as SEQ ID NO: 1, and if the 97 th position is G and the 232 th position is C, the goat is judged to be Guizhou black goat; if the sequence of the amplification product of the black goat to be identified is shown as SEQ ID NO: and 1, if the 97 th position is T and the 232 th position is T, the goat is judged to be le to black.
5. The molecular assay method of claim 4, wherein the 25 μ L PCR amplification reaction system comprises: PCR mixture 12. mu.L, template 2. mu.L of upstream and downstream primers 1. mu. L, cDNA, respectively, and double distilled water 9. mu.L.
6. The molecular assay method of claim 4, wherein the PCR amplification reaction procedure comprises: pre-denaturation at 95 ℃ for 5 min; at 94 ℃ for 40s, at 53 ℃ for 40s, at 72 ℃ for 50s, for 35 cycles; extension at 72 ℃ for 10 min.
7. Use of the molecular marker of claim 1 or the primer pair of claim 2 or the kit of claim 3 or the molecular detection method of claim 4 for identifying Guizhou black goat and le-to-black goat.
8. Use of the molecular marker of claim 1 or the primer pair of claim 2 or the kit of claim 3 or the molecular assay of claim 4 for pure breeding and selective breeding of black goats and/or black goats in Guizhou province.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111248668.4A CN113774153B (en) | 2021-10-26 | 2021-10-26 | Molecular marker for identifying Guizhou black goat and le-Zhi black goat, detection method and application |
| PCT/CN2021/137318 WO2023070857A1 (en) | 2021-10-26 | 2021-12-13 | Molecular marker for distinguishing guizhou black goat from lezhi black goat, and detection method therefor and use thereof |
| ZA2023/00360A ZA202300360B (en) | 2021-10-26 | 2023-01-09 | Molecular marker for identifying guizhou black goat and lezhi black goat, detection method and application thereof |
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| CN202111248668.4A CN113774153B (en) | 2021-10-26 | 2021-10-26 | Molecular marker for identifying Guizhou black goat and le-Zhi black goat, detection method and application |
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| CN113774153B CN113774153B (en) | 2023-02-17 |
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| WO (1) | WO2023070857A1 (en) |
| ZA (1) | ZA202300360B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023070857A1 (en) * | 2021-10-26 | 2023-05-04 | 贵州省畜牧兽医研究所 | Molecular marker for distinguishing guizhou black goat from lezhi black goat, and detection method therefor and use thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116970712B (en) * | 2023-09-04 | 2024-03-22 | 湖北省农业科学院畜牧兽医研究所 | SNP molecular marker related to reproduction traits on goat chromosome 13 and application |
| CN116904615B (en) * | 2023-09-04 | 2024-02-02 | 湖北省农业科学院畜牧兽医研究所 | SNP molecular marker related to reproduction traits on goat chromosome 20 and application |
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| CN101029341A (en) * | 2007-04-11 | 2007-09-05 | 中国农业科学院北京畜牧兽医研究所 | Method for screening high-reproduction goat and its special primer |
| US20110129825A1 (en) * | 2007-08-03 | 2011-06-02 | Melba Stinnett Ketchum | Compositions, methods and systems for the simultaneous determination of parentage, identity, sex, genotype and/or phenotype and breed determination in animals |
| CN103202267A (en) * | 2013-04-11 | 2013-07-17 | 贵州省畜牧兽医研究所 | Selective breeding method for improving meat performance of black goats of Guizhou |
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| CN104488815A (en) * | 2014-12-02 | 2015-04-08 | 贵州由由农业开发有限公司 | Cultivating method of 'internal three-element' commercial mutton |
| CN104938417B (en) * | 2015-06-29 | 2017-11-03 | 四川省畜牧科学研究院 | A kind of breeding method of the plump lamb type meat Goats Breeds in another name for Sichuan Province |
| CN113774153B (en) * | 2021-10-26 | 2023-02-17 | 贵州省畜牧兽医研究所 | Molecular marker for identifying Guizhou black goat and le-Zhi black goat, detection method and application |
-
2021
- 2021-10-26 CN CN202111248668.4A patent/CN113774153B/en active Active
- 2021-12-13 WO PCT/CN2021/137318 patent/WO2023070857A1/en unknown
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2023
- 2023-01-09 ZA ZA2023/00360A patent/ZA202300360B/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101029341A (en) * | 2007-04-11 | 2007-09-05 | 中国农业科学院北京畜牧兽医研究所 | Method for screening high-reproduction goat and its special primer |
| US20110129825A1 (en) * | 2007-08-03 | 2011-06-02 | Melba Stinnett Ketchum | Compositions, methods and systems for the simultaneous determination of parentage, identity, sex, genotype and/or phenotype and breed determination in animals |
| CN103202267A (en) * | 2013-04-11 | 2013-07-17 | 贵州省畜牧兽医研究所 | Selective breeding method for improving meat performance of black goats of Guizhou |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023070857A1 (en) * | 2021-10-26 | 2023-05-04 | 贵州省畜牧兽医研究所 | Molecular marker for distinguishing guizhou black goat from lezhi black goat, and detection method therefor and use thereof |
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| WO2023070857A1 (en) | 2023-05-04 |
| CN113774153B (en) | 2023-02-17 |
| ZA202300360B (en) | 2023-08-30 |
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