CN114941035B - Molecular marker related to sheep stage weight character and application thereof - Google Patents
Molecular marker related to sheep stage weight character and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker related to sheep stage weight character and application thereof. The nucleotide sequence of the molecular marker related to sheep stage weight is shown as SEQ ID NO.1, wherein Y at 216bp represents T or C, and the mutation leads to T/C polymorphism of the molecular marker. According to the invention, through PCR amplification and sequence analysis of sheep OSMR genes, genes of 375 Hu sheep, 82 Dupoaching sheep and 31 Tan sheep are detected and a least square model is established, and correlation analysis is carried out on genotype and stage body weight characteristics, a T/C polymorphism site exists at the 216 th site of an amplified fragment sequence shown as SEQ ID NO.1, and finally, the amplified OSMR gene fragment can be used as a molecular marker related to sheep stage body weight. The invention can be used for selecting the TT homozygous Hu sheep entering the core group as a breeding sheep by detecting the molecular marker, and is beneficial to improving the production efficiency by using the weight of sheep in stages.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to an OSMR gene fragment serving as a molecular marker related to weight traits in sheep stages and application thereof.
Background
In the production practice of mutton sheep, the Body Weight (BW) reflects the meat producing capacity of sheep, which is an important economic index for measuring the production performance of mutton sheep, and research shows that the Body weight belongs to moderate genetic transmission (0.35-0.46), is controlled by heredity and can be effectively improved by selection; however, fewer candidate genes are reported for weight-related sheep stage.
The signaling oncostatin M receptor (oncostatin M receptor, OSMR) is a member of the interleukin-6 factor family (IL-6), and (Hui,W.,et al.,Oncostatin M(OSM)stimulates resorption and inhibits synthesis of proteoglycan in porcine articular cartilage explants.1996.495-500.)., which is closely related to cell growth, proliferation and differentiation, widely exists in different types of cells and plays an important role in body development, is mainly expressed in fat, and is involved in regulating differentiation and development of adipose tissue homeostasis (Elks,C.M.,et al.,Loss of Oncostatin M Signaling in Adipocytes Induces Insulin Resistance and Adipose Tissue Inflammation in Vivo.2016,17066-17076.). adipocytes, which can affect body lipid production and development, and thus its stage body weight. The body weight in the stage belongs to moderate genetic force and is influenced by various factors such as basic metabolism, the environment in which the body weight is positioned, feed efficiency, organism activities, feed nutrition and the like. However, it is not clear whether the OSMR gene is involved in sheep stage body weight.
The invention discusses the relevance of different genotypes and sheep stage weights by sequencing and analyzing OSMR genes, and aims to provide gene materials for the aspect of genetic improvement for improving sheep stage weights and accelerate the cultivation process of novel high-yield high-quality mutton sheep varieties with independent intellectual property rights.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a molecular marker related to sheep stage weight and application thereof. The molecular marker is amplified from sheep OSMR gene, and the nucleotide sequence is shown as SEQ ID NO. 1. The method comprises the steps of amplifying sheep OSMR gene sequences through a large number of experiments, sequencing, searching polymorphic sites of the OSMR genes, analyzing the weight correlation between different genotypes and sheep stages, establishing a detection method of the polymorphic sites, and applying the molecular marker to the cultivation of high-yield high-quality mutton sheep new varieties.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in one aspect of the present invention, a molecular marker related to sheep stage body weight traits is provided, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein Y at position 216bp represents T or C, and the T/C mutation at position 216 of the sequence causes the T/C polymorphism of sheep OSMR gene at the position.
In a second aspect of the present invention there is provided a set of primers for detecting a molecular marker as defined above, any primer capable of specifically amplifying a molecular marker of the present invention or a fragment comprising a polymorphic site as defined above being suitable for detecting the molecular marker, preferably comprising forward and reverse primers having nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO. 3.
In addition, the invention also provides a set of KASPar primer groups for detecting the molecular markers, which comprises forward primers for detecting AlleleC, forward primers for detecting AlleleT 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.
In a third aspect, the present invention provides a kit for detecting the molecular marker, wherein the detection kit comprises the primer set or the KASPar primer set.
According to a fourth aspect of the present invention, there is provided a method for detecting molecular markers associated with weight traits in sheep, as described above, the method comprising detecting genomic DNA of sheep using the primer set or kit described above, the method comprising the steps of:
1) Amplifying genomic DNA of sheep using the primer pair, the KASPar primer pair or a kit comprising the primer pair;
2) And (3) identifying the polymorphic site of the amplification product obtained in the step (1).
Wherein, in step 2), the method of typing identification includes, but is not limited to, a direct sequencing method, a probe method, a gene chip method or a high resolution dissolution profile method.
A method of detecting a molecular marker for a sheep-stage body weight-related trait, KASPar comprising the steps of:
a) Extracting genome DNA from sheep blood as a sample, and performing high-throughput 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 amplification, fluorescence signals were detected using BMG PHERAstar instrument and the typing results were checked.
The fifth aspect of the present invention provides an application of the detection method of molecular markers, primer pairs or kit described above in detection of weight-related traits in sheep stage, by detecting the molecular markers provided by the present invention in genomic DNA of sheep to be detected and analyzing the type of polymorphic sites, the weight-related traits in sheep stage can be determined, and sheep with heavy weight stage can be screened out.
The sixth aspect of the present invention provides an 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 genomic DNA of sheep to determine the genotype of an OSMR gene of a sample to be detected, so that a high-yield sheep variety can be bred therefrom.
Finding mutation sites of genes, finding the relationship between genes and traits through association analysis between the genes and the traits is an important means for researching gene functions and is also a basis for marker-assisted selection.
The invention discovers that a T/C polymorphism site exists at the 216 th position of an amplified fragment by carrying out PCR amplification and sequencing on OSMR genes of sheep representing hu sheep, and determines a molecular marker related to weight of sheep at 80-180 days old stage by detecting and detecting polymorphism of 375 hu sheep, 82 Dupoaching sheep and 31 beach sheep and establishing a least square model.
The invention has the beneficial effects that:
The invention provides a molecular marker related to sheep stage weight character, in particular to a polymorphism site of T/C at the 216 th site of SEQ ID NO.1 fragment, which provides an effective detection means for breeding fast-growing sheep. The invention can be used for selecting the sheep with the TT homozygous gene as a breeding sheep through detecting the molecular marker related to the weight character of the sheep stage and the polymorphism site, so as to shorten the breeding period, improve the growth character of the sheep and be beneficial to improving the economic benefit of sheep breeding industry.
Drawings
FIG. 1 is a gel electrophoresis diagram of sheep OSMR gene fragment as a molecular marker.
FIG. 2 shows the sequencing result of mutation sites of sheep OSMR gene in the invention.
FIG. 3 shows the typing result of mutation site KASPAR SNP of sheep OSMR gene g.2443T > C in the present invention.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
Unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, all reagents used in the methods are of analytical purity or above.
Example 1 amplification of OSMR Gene
A pair of primers was designed using oligo7.0 software using sheep OSMR gene DNA (GenBank accession number: nc_ 040267.1) as a template: forward and reverse primers, primer sequences were as follows:
Forward primer (SEQ ID No. 2): 5'-TAACATCATCAGGGCATCGAG-3' A
Reverse primer (SEQ ID NO. 3): 5'-TCTGTTGTCAAATGTTGCCTA-3' A
(2) Amplification and sequencing of OSMR Gene
Genomic DNA extracted from whole blood cells of sheep was used as a DNA template. Extraction can be performed using a DNA advance kit. The total volume of the PCR reaction was 35. Mu.L, 2X PCR 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), 14. Mu.L of ddH 2 O and 1.5. Mu.L of DNA template.
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, cycling for 35 times, and extension at 72℃for 10min.
The reaction products of the above PCR amplification were detected by 1.5% agarose gel electrophoresis, and the results are shown in FIG. 1, in which lanes 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, a polymorphic site exists in the fragment, specifically Y at the 216bp site is T or C, namely the amplified OSMR gene fragment (SEQ ID NO. 1) has T/C polymorphism at the 216bp site. The peak map obtained by sequencing is subjected to bimodal detection, a mutation site is identified, the result is shown in figure 2, a 381bp fragment is obtained, and the double peaks appear, and the site is positioned at g.2443 of an OSMR gene.
Wherein SEQ ID NO.1 is shown below ：TAACATCATCAGGGCATCGAGCCAGTTTTGAAGAGGTTGGCAACACACCCATGTTCTTGGGAAGCAAGTGACTGATTAAACTGCTTCTCATGGGGATTATTTGCAGTGGGGAAACTTCCCAGGAATAAATACTTCAGTAATGACTGAATAAGAAGCACTGATGAAGAGAGACAGATTTTTCAAAATCCGTGCTTTTACTACACATGTAGTATTTAYACTGAGGGATGTTCAAAGGCCATCTTCTTTTTGCAAAGAGACAAGATTTGAATGAGAAGTTCTACAAAAAAGTGGGTGATTTTTCTTTTCTTTCCTCCCATTTTCCCCCCAGAGAAGGAATGTCAGTGAAAGACATGTATGAATTAGGCAACATTTGACAACAGA.
Sequence homology search identification:
The DNA sequence obtained after sequencing was compared for sequence homology with known physiological functional genes published in 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 shows that the homology of the sequenced sequence with partial sequence of Hu sheep OSMR gene DNA (GenBank accession number: NC_ 040267.1) reaches 99%.
Example 2 establishment of genotyping assay
1. Primer sequence design
A KASPar primer pair was designed for the C/T polymorphic site shown in the amplified fragment SEQ ID NO.1 of example 1, so as to be used for the specific detection of the polymorphic site, and the nucleotide sequence of the designed KASPar primer pair was:
Forward primer A1 (SEQ ID No. 4) for detection AlleleC: GAAGGTGACCAAGTTCATGCTATGGCCTTTGAACATCCCTCAGTA;
Forward primer A2 (SEQ ID No. 5) for detection AlleleT: GAAGGTCGGAGTCAACGGATTGGCCTTTGAACATCCCTCAGTG;
Universal reverse primer C (as set forth in SEQ ID No. 6):
AGATTTTTCAAAATCCGTGCTTTTACTACAC。
The primers are entrusted to be synthesized by Beijing Biotechnology, inc. Each set of primers in the KASPar primer pair was diluted to 10. Mu. Mol/L and mixed in a volume ratio of 12:12:30 (primer A1: primer A2: primer C) for further use.
2. DNA quality control
The whole blood of sheep is extracted with genome DNA and may be extracted with DNA extracting kit. The quality detection of the extracted genome DNA is carried out, 1% agarose electrophoresis and Nanodrop2100 are adopted for detection respectively, and the qualified DNA is required to reach: agarose electrophoresis showed single DNA bands without significant dispersion; nanodrop2100 detection A260/280 is between 1.8-2.0; there is no significant light absorption at A270 nm. The concentration of the diluted DNA template is diluted to 10-20 ng/. Mu.L for standby.
3. Genotyping assays
(1) Firstly, a K-pette liquid-separating workstation is used for respectively adding 1.5 mu L of diluted DNA template to be detected (10-20 ng/muL) and blank control (No template control, NTC, sterilized water is adopted) into 384-hole reaction plates, and drying is carried out for 30min at 60 ℃ (a drying oven, LGC company) so that the DNA is changed into dry powder for standby.
(2) Each of the above KASPar primer pairs was diluted to 10. Mu. Mol/L and was used as forward primer A1: forward primer A2: the universal reverse primer C is uniformly mixed according to the volume ratio of 12:12:30 to be used as a primer mixed solution for standby. (3) Under Kraken operation system, adding 1 XMaster Mix (1536 microwell plate, product number: part No. KBS-1016-011) and primer mixture into each reaction well by using a Meridian sample adding workstation, packaging the microwell plate on a Kube heat sealing instrument and a Fusion laser membrane sealing instrument in sequence immediately after Mix packaging, and performing high-flux water bath PCR amplification by using Hydrocyler.
The PCR reaction was performed in a high throughput water bath system Hydrocycler, with the following specific procedures:
Pre-denaturation at 94 ℃ for 15 min;
94 ℃,20 seconds (denaturation) -61 ℃ -55 ℃,1 minute (renaturation & extension), 10 cycles of amplification in the touchdown order, 0.6 ℃ decrease per cycle;
Amplification was continued for 26 cycles at 94℃for 20 seconds (denaturation) -55℃for 60 seconds.
(4) After amplification, a BMG PHERAstar instrument is used for detecting fluorescent signals and checking the parting condition, and the specific result is shown in fig. 3. Each dot in the figure represents a piece of material to be tested, wherein the red dot near the left side indicates that the locus is homozygous for genotype "CC"; blue dots near the right indicate that the locus is homozygous genotype "TT"; the green dots near the middle indicate that the locus is heterozygous genotype "TC" or "CT"; the black dots represent NTC (i.e. water control, not shown in fig. 3).
4. Application of molecular marker in sheep growth trait correlation analysis
The polymorphism of 375 Hu sheep, 82 Dupoachi sheep and 31 Tan sheep is detected in total, the genotype is determined, a least squares model is established as described below, and the correlation analysis of the genotype and the stage weight is carried out.
Yijkl＝μ+Genotypei+Pj+Ml+εijkl
Wherein Y ijkl is the observed value of the phase body weight, mu is the overall average, genotype i is the genotype effect, P j is the batch effect, M l is the variety effect, epsilon ijkl is the random error, and epsilon ijkl is assumed to be independent of each other and obey the N (0, sigma 2) distribution.
Determination of stage body weight in this example times 80d,100d,120d,140d,160d and 180d. Genotype detection results showed that of 488 individuals, there were 25 CC genotypes, 105 CT genotypes, 355 TT genotypes, and 3 individuals that were not successfully typed. The results of genotype-trait association analysis are shown in Table 1, where BW represents the body weight in kg. BW80d represents 80 day old body weight; BW100d represents body weight at 100 days of age, and so on.
TABLE 1 sheep OSMR Gene polymorphism and stage weight correlation analysis
Note that: the different letters of the subscript between the same row of data indicate significant differences (P < 0.05), and the same letter indicates insignificant differences (P > 0.05).
The results show that the T/C polymorphism site at 216bp of the nucleotide sequence shown as SEQ ID NO.1 is obviously related to sheep stage weight along with the extension of the measurement period. Wherein the phase body weight of TT genotype individuals is significantly higher than those of CT type and CC type (P < 0.05). From this, TT genotype was the dominant genotype and T allele was the dominant allele, indicating that the OSMR g.2443T > C mutation site could be used as a potential molecular marker (P < 0.05) affecting sheep stage body weight. The TT genotype is selected for seed conservation during breeding, and is used as a breeding sheep to hybridize with other sheep during breeding, so that the production efficiency of offspring sheep can be effectively improved.
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
agatttttca aaatccgtgc ttttactaca c 31
Claims (9)
1. The application of a molecular marker related to sheep stage weight traits in sheep breeding is characterized in that the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, wherein Y at 216bp represents T or C, and the mutation leads to a T/C polymorphism of the molecular marker; wherein, the phase weight of TT genotype individuals is significantly higher than CT type and CC type individuals.
2. The application of the primer group for detecting the molecular marker related to sheep stage weight characters in sheep breeding is characterized in that the primer group comprises a forward primer and a reverse primer with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO. 3; the nucleotide sequence of the molecular marker related to sheep stage weight traits is shown in SEQ ID NO.1, wherein Y at 216bp represents T or C, and the mutation leads to a T/C polymorphism of the molecular marker; wherein, the phase weight of TT genotype individuals is significantly higher than CT type and CC type individuals.
3. Use of a KASPar primer set for detecting a molecular marker associated with weight traits in sheep stage in sheep breeding, characterized in that the KASPar primer set comprises a forward primer for detecting AlleleC, a forward primer for detecting AlleleT and a universal reverse primer as shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6; the nucleotide sequence of the molecular marker related to sheep stage weight traits is shown in SEQ ID NO.1, wherein Y at 216bp represents T or C, and the mutation leads to a T/C polymorphism of the molecular marker; wherein, the phase weight of TT genotype individuals is significantly higher than CT type and CC type individuals.
4. The application of the kit for detecting the molecular marker related to the weight character of sheep stage in sheep breeding is characterized in that the kit comprises a primer pair with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3 or a KASPar primer pair with nucleotide sequences shown as SEQ ID NO. 4-6.
5. Use of a method for detecting molecular markers associated with sheep stage body weight traits in sheep breeding comprising the steps of:
1) Amplifying the Hu sheep genome DNA by using a PCR primer group with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3 or a KASPar primer group with nucleotide sequences shown as SEQ ID NO.4-6 or a detection kit containing the PCR primer group or the KASPar primer group;
2) Carrying out typing identification on the site of the T or C represented by Y at 216bp of the amplification product obtained in the step 1) as shown in SEQ ID NO. 1;
the phase body weight of TT genotype individuals is significantly higher than those of CT and CC types.
6. The method according to claim 5, wherein the typing method in step 2) is a direct sequencing method, a fluorescent probe method, a gene chip method or a high resolution dissolution profile method.
7. The use according to claim 5, wherein the KASPar primer pair according to claim 3 is used for amplification, and after the amplification is completed, the typing result is determined by detecting a fluorescent signal.
8. The use according to any one of claims 1 to 7, wherein the purpose of breeding is to cultivate high-yielding sheep.
9. A molecular marker related to sheep stage weight trait or a PCR primer set or a KASPar primer set for detecting the molecular marker related to sheep stage weight trait, or a kit for detecting the molecular marker related to sheep stage weight trait, or application of a detection method of the molecular marker related to sheep stage weight trait in sheep stage weight trait related detection, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein Y at 216bp represents T or C, and the mutation leads to T/C polymorphism of the molecular marker; wherein, the phase weight of TT genotype individuals is significantly higher than CT type and CC type individuals; the nucleotide sequences of the PCR primer group are shown as SEQ ID NO.2 and SEQ ID NO. 3; the nucleotide sequences of the KASPar primer group are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
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