CN114941035A - Molecular marker related to sheep stage weight traits and application thereof - Google Patents
Molecular marker related to sheep stage weight traits and application thereof Download PDFInfo
- Publication number
- CN114941035A CN114941035A CN202210697898.7A CN202210697898A CN114941035A CN 114941035 A CN114941035 A CN 114941035A CN 202210697898 A CN202210697898 A CN 202210697898A CN 114941035 A CN114941035 A CN 114941035A
- Authority
- CN
- China
- Prior art keywords
- sheep
- molecular marker
- primer
- seq
- detecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241001494479 Pecora Species 0.000 title claims abstract description 81
- 239000003147 molecular marker Substances 0.000 title claims abstract description 39
- 238000009395 breeding Methods 0.000 claims abstract description 16
- 230000001488 breeding effect Effects 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 230000035772 mutation Effects 0.000 claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 108020004414 DNA Proteins 0.000 claims description 27
- 238000001514 detection method Methods 0.000 claims description 15
- TWQHGBJNKVFWIU-UHFFFAOYSA-N 8-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-8-azaspiro[4.5]decane-7,9-dione Chemical compound C1C(=O)N(CCCCN2CCN(CC2)C=2N=C3C=CC=CC3=CC=2)C(=O)CC21CCCC2 TWQHGBJNKVFWIU-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 238000012163 sequencing technique Methods 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 6
- 238000002844 melting Methods 0.000 claims description 2
- 230000008018 melting Effects 0.000 claims description 2
- 101150080012 OSMR gene Proteins 0.000 abstract description 19
- 230000037396 body weight Effects 0.000 abstract description 17
- 239000012634 fragment Substances 0.000 abstract description 13
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000012408 PCR amplification Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000010219 correlation analysis Methods 0.000 abstract description 3
- 101000586302 Homo sapiens Oncostatin-M-specific receptor subunit beta Proteins 0.000 abstract 1
- 238000012300 Sequence Analysis Methods 0.000 abstract 1
- 108010082522 Oncostatin M Receptors Proteins 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 102000004140 Oncostatin M Human genes 0.000 description 3
- 108090000630 Oncostatin M Proteins 0.000 description 3
- 102000003987 Oncostatin M Receptors Human genes 0.000 description 3
- 238000012098 association analyses Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000002869 basic local alignment search tool Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241001386813 Kraken Species 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000037081 physical activity Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000030968 tissue homeostasis Effects 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/6876—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
-
- 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
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a molecular marker related to sheep stage weight traits and application thereof. The nucleotide sequence of the molecular marker related to the sheep stage body weight is shown as SEQ ID NO.1, wherein Y at 216bp represents T or C, and the mutation causes T/C polymorphism of the molecular marker. The invention carries out PCR amplification and sequence analysis on sheep OSMR genes, detects the genes of 375 Hu sheep, 82 DuPo sheep and 31 Tan sheep, establishes a least square model, carries out correlation analysis on the characteristics of the genotype and the phase weight, finds that a T/C polymorphic site exists at the 216 th site of the sequence of an amplified fragment shown as SEQ ID NO.1, and finally determines that the amplified OSMR gene fragment can be used as a molecular marker related to the sheep phase weight. The invention can be used for selecting and reserving the core group of the Hu sheep with TT homozygote as the breeding sheep by detecting the molecular marker, and is beneficial to improving the production efficiency by taking the stage weight of the sheep.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to an OSMR gene fragment as a molecular marker related to the influence on the weight traits of sheep in the sheep stage and application thereof.
Background
In the production practice of mutton sheep, the Body Weight (BW) reflects the meat production capacity of sheep, and is an important economic index for measuring the production performance of mutton sheep, and researches show that the Body weight in the stage belongs to medium heritability (0.35-0.46), is genetically controlled and can be effectively improved through selection; however, few candidate genes related to the sheep stage body weight are reported.
The signaling Oncostatin M receptor (OSMR) is a member of the interleukin-6 factor family (IL-6) and is closely related to the growth, proliferation and differentiation of cells (Hui, W., et al, Oncostatin M (OSM) peptides restriction and inhibition synthesis of protein in protein tissue extensions.1996.495-500.). It is expressed mainly in fat and is involved in the regulation of Adipose Tissue homeostasis (Elks, C.M., et al, Loss of Oncostatin M Signaling in Adipocytes indulges in vivo 2016, 17066. 17076.). Differentiation and development of adipocytes can affect the production and development of body lipids, and thus, their stage body weight. Stage weight is moderate heritability, and is affected by multiple factors such as basal metabolism, environment, feed efficiency, body activity, and feed nutrition. However, it is not clear whether the OSMR gene is related to the sheep stage body weight.
The invention discusses the relevance of different genotypes of the OSMR genes and sheep stage weight by sequencing and analyzing the OSMR genes, aims to provide gene materials for improving the genetic improvement aspect of the sheep stage weight and accelerates the breeding process of high-yield high-quality new mutton sheep varieties with independent intellectual property rights.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a molecular marker related to sheep stage body weight and application thereof. The molecular marker is obtained by amplifying sheep OSMR gene, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1. The sheep OSMR gene sequence is amplified and sequenced through a large number of experiments, the polymorphic sites of the OSMR gene are searched, the correlation between different genotypes and sheep stage weight is analyzed, the detection method of the polymorphic sites is established, and the molecular marker is applied to the cultivation of new varieties of high-yield high-quality mutton sheep.
In order to achieve the purpose, the invention adopts the following technical scheme that:
one aspect of the invention provides a molecular marker related to sheep phase body weight traits, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein Y at the 216bp position represents T or C, and T/C polymorphism of sheep OSMR gene at the position is caused due to T/C mutation of the sequence at the 216 base.
In a second aspect of the present invention, there is provided a primer set for detecting the above molecular marker, and any primer capable of specifically amplifying the molecular marker of the present invention or a fragment containing the above polymorphic site is suitable for detecting the molecular marker, and preferably, the primer set comprises a forward primer and a reverse primer having nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.
In addition, the invention also provides a group of KASPar primer groups for detecting the molecular markers, which comprise forward primers, forward primers and universal reverse primers, wherein the nucleotide sequences of the forward primers are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the forward primers are used for detecting AlleLET.
The third aspect of the present invention provides a kit for detecting the above molecular marker, wherein the kit comprises the above primer set or KASPar primer set.
The fourth aspect of the invention provides a detection method of a molecular marker related to the sheep stage weight trait, wherein the molecular marker is as described above, the method comprises the step of detecting sheep genomic DNA by using the primer group or the kit, and the specific detection method comprises the following steps:
1) amplifying the genomic DNA of the sheep by using the primer pair, the KASPar primer pair or the kit containing the primer pair;
2) identifying the polymorphic sites of the amplification products obtained in step 1).
In step 2), the typing and identification methods include, but are not limited to, direct sequencing, probe, gene chip, or high resolution melting curve.
The KASPar method for detecting the molecular marker of the sheep stage weight-related traits comprises the following steps:
a) extracting genome DNA by using sheep blood as a sample, and performing high-flux water bath PCR amplification on the extracted genome DNA by using KASPar primers with nucleotide sequences shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6;
b) after the amplification is finished, a BMG PHERAStar instrument is used for detecting a fluorescence signal and checking a typing result.
The fifth aspect of the invention provides the application of the detection method of the molecular marker, the primer pair or the kit in the detection of the sheep stage weight-related traits, wherein the sheep stage weight high-low traits can be determined by detecting the molecular marker provided by the invention in the genomic DNA of the sheep to be detected and analyzing the type of the polymorphic site, so that the sheep with large stage weight can be screened.
The sixth aspect of the invention provides the application of the detection method of the molecular marker, the primer pair or the kit in sheep breeding, wherein the primer pair or the kit is used for amplifying and detecting sheep genomic DNA to determine the genotype of the OSMR gene of a sample to be detected, so that a high-yield sheep variety can be bred from the sample.
The method is an important means for researching gene functions and is also a basis for carrying out marker-assisted selection by searching mutation sites of genes and discovering the relationship between the genes and traits through association analysis between the genes and the traits.
The invention discovers that a T/C polymorphic site exists at the 216 th site of an amplified fragment by carrying out PCR amplification and sequencing on the OSMR gene of a representative sheep variety Hu sheep, detects the polymorphism of 375 Hu sheep, 82 Du Po sheep and 31 Tan sheep and establishes a least square model, and determines a molecular marker related to the weight of the sheep at the age of 80-180 days, wherein the molecular marker can be used for breeding high-yield sheep and breeding new varieties of high-yield high-quality mutton sheep, provides an effective genetic engineering means for genetic improvement of the weight of the sheep at the stage, and has great practical application value.
The invention has the beneficial effects that:
the invention provides a molecular marker related to sheep stage weight traits, in particular to a polymorphic site of the 216 th T/C of a SEQ ID NO.1 fragment, and provides an effective detection means for breeding of fast growing sheep. The invention can be used for selecting sheep homozygous for TT as breeding sheep by detecting molecular markers related to the weight traits of sheep in the sheep stage and causing the polymorphic sites, can be used for breeding, shortens the breeding period, is used for improving the growth traits of sheep, and is beneficial to improving the economic benefit of sheep breeding industry.
Drawings
FIG. 1 is a gel electrophoresis image of an OSMR gene fragment of sheep used as a molecular marker.
FIG. 2 is the sequencing result of the mutation site of the sheep OSMR gene in the present invention.
FIG. 3 shows the result of KASPar SNP typing of g.2443T > C mutation site of sheep OSMR gene in the present invention.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention.
Unless otherwise specified, the techniques used in the examples are conventional and well known to those skilled in the art, and unless otherwise specified, the reagents used in the methods are analytical grade or above.
Example 1 amplification of OSMR Gene
A pair of primers was designed using goat OSMR gene DNA (GenBank accession No.: NC-040267.1) as a template and Oligo7.0 software: a forward primer and a reverse primer, wherein the primer sequences are as follows:
forward primer (SEQ ID NO. 2): 5'-TAACATCATCAGGGCATCGAG-3'
Reverse primer (SEQ ID NO. 3): 5'-TCTGTTGTCAAATGTTGCCTA-3'
(2) Amplification and sequencing of OSMR genes
Genomic DNA extracted from whole blood cells of sheep was used as a DNA template. Extraction can be performed with a DNA Advance kit. The total volume of PCR reaction was 35. mu.L, 2 XPCR Master Mix 17.5. mu.L, 1. mu.L of forward primer (10. mu. mol/L), 1. mu.L of reverse primer (10. mu. mol/L), ddH 2 O14. mu.L, DNA template 1.5. mu.L.
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 52.8 ℃ for 30s, extension at 72 ℃ for 30s, and circulation for 35 times, and finally extension at 72 ℃ for 10 min.
The reaction product of the above PCR amplification was detected by 1.5% agarose gel electrophoresis, and the results are shown in FIG. 1, in which lane M: DL 2000Marker, lanes 1-10: results of OSMR gene amplification. Sequencing the PCR fragment obtained by amplification, wherein the specific nucleotide sequence of the amplified fragment is shown as SEQ ID NO.1, wherein a polymorphic site exists in the fragment, particularly Y at the 216bp site is T or C, namely T/C polymorphism exists at the 216bp site of the amplified OSMR gene fragment (SEQ ID NO. 1). And (3) carrying out double peak detection on a peak image obtained by sequencing, identifying a mutation site, and obtaining a 381bp fragment and generating double peaks as shown in a graph 2, wherein the site is positioned at g.2443 of the OSMR gene.
Wherein SEQ ID NO.1 is as follows: TAACATCATCAGGGCATCGAGCCAGTTTTGAAGAGGTTGGCAACACACCCATGTTCTTGGGAAGCAAGTGACTGATTAAACTGCTTCTCATGGGGATTATTTGCAGTGGGGAAACTTCCCAGGAATAAATACTTCAGTAATGACTGAATAAGAAGCACTGATGAAGAGAGACAGATTTTTCAAAATCCGTGCTTTTACTACACATGTAGTATTTAYACTGAGGGATGTTCAAAGGCCATCTTCTTTTTGCAAAGAGACAAGATTTGAATGAGAAGTTCTACAAAAAAGTGGGTGATTTTTCTTTTCTTTCCTCCCATTTTCCCCCCAGAGAAGGAATGTCAGTGAAAGACATGTATGAATTAGGCAACATTTGACAACAGA are provided.
And (3) carrying out sequence homology search identification:
the DNA sequence obtained after sequencing was compared for sequence homology with known physiological functional genes published in the GenBank database by BLAST (basic Local Alignment Search tool) software of the National Center for Biotechnology Information (NCBI, National Center for Biotechnology Information, http:// www.ncbi.nlm.nih.gov) website to identify and obtain functional Information of the DNA sequence. The search result showed that the homology of the sequenced sequence with the Hu sheep OSMR gene DNA (GenBank accession number: NC-040267.1) was 99%.
Example 2 establishment of genotyping assay
1. Primer sequence design
Designing KASPar primer pair aiming at the C/T polymorphic site shown in the amplified fragment SEQ ID NO.1 in example 1 so as to be used for the specific detection of the polymorphic site, wherein the nucleotide sequence of the designed KASPar primer pair is as follows:
forward primer A1(SEQ ID NO.4) for detecting AlleC: GAAGGTGACCAAGTTCATGCTATGGCCTTTGAACATCCCTCAGTA, respectively;
forward primer A2(SEQ ID NO.5) for detecting AlleleT: GAAGGTCGGAGTCAACGGATTGGCCTTTGAACATCCCTCAGTG, respectively;
a universal reverse primer C (shown as SEQ ID NO. 6):
AGATTTTTCAAAATCCGTGCTTTTACTACAC。
the above primers were synthesized by Beijing Biotechnology Ltd. Each primer set in the KASPar primer pair is diluted to 10 mu mol/L and mixed according to the volume ratio of 12:12:30 (primer A1: primer A2: primer C) for later use.
2. DNA quality control
The extraction of genome DNA of sheep whole blood can be carried out by adopting a DNA extraction kit. And (3) performing quality detection on the extracted genome DNA, and respectively detecting by adopting 1% agarose electrophoresis and Nanodrop2100, wherein the qualified DNA requirements reach: agarose electrophoresis showed that the DNA band was single and not dispersed significantly; the Nanodrop2100 detects that A260/280 is between 1.8 and 2.0; a270nm had no significant light absorption. The concentration of the diluted DNA template is diluted to 10-20 ng/. mu.L for standby.
3. Genotyping assay
(1) Firstly, 1.5 muL of diluted DNA template (10-20 ng/muL) to be detected and blank control (No template control, NTC, adopting sterile water) are respectively added into a 384-hole reaction plate by using a K-pette liquid separation workstation, and the DNA is dried for 30min at 60 ℃ (a drying box, LGC company) to become dry powder for standby.
(2) Each primer of the above KASPar primer pair was diluted to 10. mu. mol/L and sequenced according to forward primer A1: forward primer a 2: the volume ratio of the universal reverse primer C is 12:12:30, and the universal reverse primer C is uniformly mixed to be used as a primer mixed solution for standby. (3) Under a Kraken operating system, a Meridian sample adding workstation is used for respectively adding 1 xMaster Mix (1536 micro-porous plate, Part No. KBS-1016-plus 011) and a primer mixed solution into each reaction hole, the micro-porous plates are sequentially placed on a Kube heat sealing instrument and a Fusion laser membrane sealing instrument for membrane sealing after the Mix split charging is finished, and the high-flux water bath PCR amplification is carried out by using a Hydrocycler.
The PCR reaction is carried out in a high-flux water bath system Hydrocycler, and the specific procedures are as follows:
pre-denaturation at 94 ℃ for 15 min;
amplification in touch down sequence for 10 cycles of 0.6 ℃ reduction per cycle at 94 ℃ for 20 seconds (denaturation) -61 ℃ to 55 ℃ for 1 minute (renaturation & elongation);
amplification was continued for 26 cycles at 94 ℃ for 20 seconds (denaturation) to 55 ℃ for 60 seconds.
(4) After the amplification is finished, a BMG PHERAStar instrument is used for detecting a fluorescence signal and checking the typing condition, and the specific result is shown in FIG. 3. Each dot in the figure represents a piece of material to be tested, with the red dot near the left indicating that the locus is of homozygous genotype "CC"; blue dots near the right indicate that the locus is homozygous genotype "TT"; the green circle near the middle indicates that the locus is a heterozygous genotype "TC" or "CT"; the black dots represent NTCs (i.e., water control, not shown in fig. 3).
4. Application of molecular marker in sheep growth trait association analysis
The test detects the polymorphism of 375 Hu sheep, 82 Du Po sheep and 31 Tan sheep, determines the genotype, establishes the least square model as described below, and performs the correlation analysis of the genotype and the stage weight.
Y ijkl ＝μ+Genotype i +P j +M l +ε ijkl
Wherein Y is ijkl Is an observed value of stage body weight, mu is the mean population, Genotype i For genotype effects, P j For batch effect, M l Being a species effect,. epsilon ijkl For random errors, assume ε ijkl Independently of each other, obey an N (0, σ 2) distribution.
In this example, the measurement time of the body weight at the stage was 80d, 100d, 120d, 140d, 160d and 180 d. Genotype testing showed that there were 25 CC genotypes, 105 CT genotypes, 355 TT genotypes, and 3 non-typed individuals in 488 individuals. The results of the genotype and trait association analysis are shown in table 1, where BW represents the stage weight in kg. BW80d represents 80 day old body weight; BW100d represents 100 days old body weight, and so on.
TABLE 1 sheep OSMR Gene polymorphism and phase body weight correlation analysis
Note: the corner marks between the data in the same row indicate significant difference (P <0.05), and the same letter indicates insignificant difference (P > 0.05).
The results show that the T/C polymorphism site at 216bp with the nucleotide sequence shown as SEQ ID NO.1 is significantly related to the sheep phase body weight along with the extension of the determination period. Wherein the stage body weight of TT genotype individuals is significantly higher than that of CT type and CC type individuals (P < 0.05). Therefore, the TT genotype is a dominant genotype, and the T allele is a dominant allele, which indicates that the OSMR g.2443T > C mutation site can be used as a potential molecular marker (P <0.05) influencing the sheep stage weight. TT genetype is selected for breed conservation during breeding, and the TT genetype is taken as a breeding sheep to be crossed with other sheep during breeding, so that the production efficiency of the offspring sheep can be effectively improved.
Sequence listing
<110> university of agriculture in Gansu province
<120> molecular marker related to sheep stage weight traits and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
taacatcatc agggcatcga gccagttttg aagaggttgg caacacaccc atgttcttgg 60
gaagcaagtg actgattaaa ctgcttctca tggggattat ttgcagtggg gaaacttccc 120
aggaataaat acttcagtaa tgactgaata agaagcactg atgaagagag acagattttt 180
caaaatccgt gcttttacta cacatgtagt atttayactg agggatgttc aaaggccatc 240
ttctttttgc aaagagacaa gatttgaatg agaagttcta caaaaaagtg ggtgattttt 300
cttttctttc ctcccatttt ccccccagag aaggaatgtc agtgaaagac atgtatgaat 360
taggcaacat ttgacaacag a 381
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
taacatcatc agggcatcga g 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tctgttgtca aatgttgcct a 21
<210> 4
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gaaggtgacc aagttcatgc tatggccttt gaacatccct cagta 45
<210> 5
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaaggtcgga gtcaacggat tggcctttga acatccctca gtg 43
<210> 6
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agatttttca aaatccgtgc ttttactaca c 31
Claims (10)
1. A molecular marker related to sheep stage weight traits is characterized in that the nucleotide sequence is shown as SEQ ID NO.1, wherein Y at 216bp represents T or C, and the mutation causes T/C polymorphism of the molecular marker.
2. A set of primers for detecting the molecular marker of claim 1, comprising a forward primer and a reverse primer having the nucleotide sequences shown in SEQ ID No.2 and SEQ ID No. 3.
3. A set of KASPar primer sets for detecting the molecular marker of claim 1, which comprises a forward primer for detecting AlleLEC, a forward primer for detecting AlleLET and a universal reverse primer, the nucleotide sequences of which are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
4. A kit for detecting the molecular marker of claim 1, comprising the primer pair of claim 2 or the KASPar primer pair of claim 3.
5. A detection method of a molecular marker related to sheep stage weight traits comprises the following steps:
1) amplifying Hu sheep genomic DNA using the PCR primer set of claim 2 or the KASPar primer set of claim 3, or using the detection kit of claim 4;
2) typing and identifying the polymorphic sites of the amplification products obtained in the step 1).
6. The detection method according to claim 5, wherein the typing and identification method in step 2) is a direct sequencing method, a fluorescence probe method, a gene chip method or a high resolution melting curve method.
7. The detection method according to claim 5, wherein the KASPar primer pair of claim 3 is used for amplification, and after the amplification is completed, the typing result is determined by detecting a fluorescent signal.
8. Use of the molecular marker of claim 1 or the primer set of claim 2 or 3, or the kit of claim 4, or the detection method of any one of claims 5 to 7 for a sheep stage weight trait-related detection.
9. Use of a molecular marker according to claim 1 or a primer pair according to claim 2 or 3, or a kit according to claim 4, or a detection method according to any one of claims 5 to 7 in sheep breeding.
10. Use according to claim 9, characterized in that the breeding is aimed at breeding high-producing sheep.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210697898.7A CN114941035A (en) | 2022-06-20 | 2022-06-20 | Molecular marker related to sheep stage weight traits and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210697898.7A CN114941035A (en) | 2022-06-20 | 2022-06-20 | Molecular marker related to sheep stage weight traits and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114941035A true CN114941035A (en) | 2022-08-26 |
Family
ID=82910865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210697898.7A Pending CN114941035A (en) | 2022-06-20 | 2022-06-20 | Molecular marker related to sheep stage weight traits and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114941035A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1361788A (en) * | 1999-05-13 | 2002-07-31 | 宾夕法尼亚州研究基金会 | Genetic Marker for meat quality, growth, carcass and reproductive traits in livestock |
| US20090291855A1 (en) * | 2008-05-23 | 2009-11-26 | Korea Institute Of Science And Technology | Method for confirming the exposure on chrysene |
| CN109913559A (en) * | 2019-03-28 | 2019-06-21 | 甘肃农业大学 | RYR2 gene is as the molecular labeling and its application for influencing sheep forage conversion ratio |
| CN113584183A (en) * | 2021-08-03 | 2021-11-02 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker influencing weaning weight traits of alpine merino sheep and application thereof |
-
2022
- 2022-06-20 CN CN202210697898.7A patent/CN114941035A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1361788A (en) * | 1999-05-13 | 2002-07-31 | 宾夕法尼亚州研究基金会 | Genetic Marker for meat quality, growth, carcass and reproductive traits in livestock |
| US20090291855A1 (en) * | 2008-05-23 | 2009-11-26 | Korea Institute Of Science And Technology | Method for confirming the exposure on chrysene |
| CN109913559A (en) * | 2019-03-28 | 2019-06-21 | 甘肃农业大学 | RYR2 gene is as the molecular labeling and its application for influencing sheep forage conversion ratio |
| CN113584183A (en) * | 2021-08-03 | 2021-11-02 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker influencing weaning weight traits of alpine merino sheep and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 佚名: "rs420893736", 《ENSEMBL》, 29 April 2022 (2022-04-29), pages 1 * |
| 张军霞;乐祥鹏;王维民;王志兰;张娜娜;胡瑞雪;谢云龙;孙武;邓颖;李发弟;: "绵羊TrkA基因的表达及其SNPs与产羔性状的关联性分析", 农业生物技术学报, no. 08, 1 August 2016 (2016-08-01), pages 52 - 60 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110791574B (en) | Molecular marker associated with goat lambing number and growth traits and application thereof | |
| CN107475412B (en) | Molecular marker related to egg-laying traits of chickens and application of molecular marker in chicken breeding | |
| CN113502335B (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN110643716B (en) | Molecular marker related to sheep tail fat weight and application thereof | |
| CN112251518B (en) | Molecular marker associated with lambing number and growth traits in goat RSAD2 gene and application thereof | |
| CN113913530B (en) | Molecular marker related to sheep body height and application thereof | |
| CN105969879B (en) | Primer group for high-throughput detection of AhFAD2A gene mutation site typing and detection method | |
| CN115029444A (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN107287283B (en) | High-throughput detection kit for multiple SNP sites related to children susceptibility diseases and use method thereof | |
| CN116083592A (en) | Molecular marker related to sheep growth traits and application thereof | |
| CN115109856A (en) | Molecular marker related to sheep stage body weight, detection method and application thereof | |
| CN115044682A (en) | Molecular marker related to growth traits of Hu sheep, detection method and application thereof | |
| CN115992251A (en) | Molecular marker related to Hu sheep growth traits and application thereof | |
| CN114941035A (en) | Molecular marker related to sheep stage weight traits and application thereof | |
| CN112226520B (en) | Method for detecting molecular marker in goat circular RNA and application thereof | |
| CN116855613B (en) | Molecular marker, primer, kit, method and application of pig carcass traits | |
| CN116676400B (en) | Molecular marker, primer, kit, method and application related to intramuscular fat traits of pigs | |
| CN117051128B (en) | NARS2 gene molecular marker related to pork quality traits and application thereof | |
| CN116083604B (en) | SNP molecular marker affecting sheep weaning weight and application thereof | |
| CN111850139B (en) | Molecular marker located on pig chromosome 12 and related to formation of pig monocrchidism and application | |
| CN114717336B (en) | Molecular marker related to sheep viscera weight and application thereof | |
| CN111808974B (en) | SNP molecular marker located on pig chromosome 9 and related to scrotal hernia and application | |
| CN115181804A (en) | Molecular marker related to sheep growth traits, detection method and application thereof | |
| CN115074449A (en) | Molecular marker related to sheep tail fat deposition and application thereof | |
| CN116064831A (en) | Molecular marker related to sheep body ruler, detection method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |