CN118621038B - SNP molecular marker related to sheep body length characters, primer set, kit and application thereof - Google Patents
SNP molecular marker related to sheep body length characters, primer set, kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of SNP molecular markers, and particularly relates to a SNP molecular marker related to sheep body length characters, a primer set, a kit and application thereof. The SNP molecular marker is positioned at 51717733bp locus on chromosome 4 of sheep genome, and the SNP molecular marker has C/A base mutation. The SNP molecular marker has obvious correlation with sheep body length characters, enriches a sheep body length molecular marker database, and researches show that the body length of adult sheep individuals with CC genotypes is obviously higher than that of sheep individuals with genotypes of CA and AA, CC homozygous individuals with adult body length advantages can be selected and remained in the sheep breeding process, particularly, individuals with body length advantages are selected and remained in the lamb stage, and the SNP molecular marker has potential application value for large-scale molecular breeding of sheep.
Description
Technical Field
The invention belongs to the technical field of SNP molecular markers, and particularly relates to a SNP molecular marker related to sheep body length characters, a primer set, a kit and application thereof.
Background
Sheep body length traits are one of the important indicators affecting sheep body size. The length of sheep is affected by genetic factors, and varies from 1.2 meters to 1.8 meters depending on the variety, and does not include tails 7.5-25 cm long. Therefore, the screening of the major genes or molecular genetic markers related to sheep body length can be used for molecular breeding of subsequent sheep, so that the sheep body length is increased, the sheep body shape is increased, the progress of the sheep body length genetic breeding is accelerated, and the increasingly prominent contradiction between the low production efficiency of the current mutton sheep and the increasing mutton demands of people is solved.
However, there is no report in the art of the correlation of sheep body length traits with the molecular markers described in the present invention.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to sheep body length characters, a primer set, a kit and application thereof, wherein the SNP molecular marker has obvious correlation with sheep body length characters, and enriches a sheep body length molecular marker database.
The invention provides an SNP molecular marker related to sheep body length characters, wherein the SNP molecular marker is positioned at 51717733bp locus on chromosome 4 of sheep genome, and the SNP molecular marker has C/A base mutation.
The invention also provides a primer set for detecting the SNP molecular marker in the technical scheme, wherein the primer set comprises an upstream primer, a downstream primer and an extension primer; the nucleotide sequences of the upstream primer, the downstream primer and the extension primer are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
The invention also provides a kit which comprises the primer set and the PCR amplification reagent.
Preferably, the PCR amplification reagent comprises dNTPs, taq DNA polymerase, mgCl 2, PCR reaction buffer, shrimp alkaline phosphatase and iplex reaction reagent;
The kit also includes standard positive template DNA.
The invention also provides the SNP molecular marker of the technical scheme, the primer group of the technical scheme or the application of the kit of the technical scheme in sheep body length molecular marker assisted breeding.
Preferably, the sheep body length molecular marker assisted breeding comprises prediction of the dominance of adult sheep body length traits.
The invention also provides a method for predicting whether the adult length of sheep has advantages, which comprises the following steps:
Taking genome DNA of sheep to be detected as a template, and performing PCR amplification reaction by using an upstream primer and a downstream primer in the primer set in the technical scheme to obtain a PCR amplification product;
performing digestion reaction on the PCR amplification product to obtain a digested PCR amplification product;
taking the digested PCR amplification product as a template, and performing an extension reaction by using extension primers in the primer set in the technical scheme to obtain an extension product;
genotyping the extension product to obtain a genotype result;
predicting the adult length of the sheep to be tested according to the genotype result: when the genotype of 51717733bp locus on chromosome 4 of sheep genome is CC genotype, the adult postcorpora length of the sheep to be detected is long, and the sheep has the advantage of corpora length;
When the genotype of 51717733bp locus on chromosome 4 of sheep genome is CA or AA genotype, the adult length of sheep to be detected is short, and the sheep has no length advantage.
Preferably, the reaction system of the PCR amplification reaction comprises the following components in terms of 5 mu L: 20-50 ng/mu L of genomic DNA 1 mu L, PCR reaction buffer solution 0.5 mu L, 25mmol/L MgCl 2 0.4 mu L, 25 mu mol/L dNTPs 0.1 mu L, 0.5 mu mol/L PCR primer mixture 1 mu L, 5U/mu L Taq DNA polymerase 0.2 mu L and deionized water 1.8 mu L;
The PCR amplification reaction is performed at 95 ℃ for 2min in a pre-denaturation mode; denaturation at 95℃for 30s, annealing at 56℃for 30s,
Extending at 72 ℃ for 60s, and 45 cycles are total; maintained at 72℃for 5min.
Preferably, the digestion system of the digestion reaction comprises, in terms of 2 μl: shrimp alkaline phosphatase Buffer 0.17 [ mu ] L, shrimp alkaline phosphatase 0.3 [ mu ] L of 1.7U/[ mu ] L and deionized water 1.53 [ mu ] L;
The digestion reaction is performed by the following steps: 40min at 37℃and 5min at 85 ℃.
Preferably, the extension system of the extension reaction comprises, in terms of 2 μl: iplex Buffer Plus 0.2.2 [ mu ] L, iplex Terminator 0.2 [ mu ] L, extension primer 0.94 [ mu ] L, iplex enzyme 0.041 [ mu ] L and deionized water 0.619 [ mu ] L;
the extension reaction is carried out by the following steps: 94 ℃ for 30s;
[94℃for 5s, (52℃for 5s,80℃for 5 s) ], wherein said (52℃for 5s,80℃for 5 s) is performed for 5 cycles, and said [94℃for 5s, (52℃for 5s,80℃for 5 s) ] is performed for 40 cycles; and 3min at 72 ℃.
The beneficial effects are that:
The invention provides an SNP molecular marker related to sheep body length characters, wherein the SNP molecular marker is positioned at 51717733bp locus on chromosome 4 of sheep genome, and the SNP molecular marker has C/A base mutation. The SNP molecular marker has obvious correlation with sheep body length characters, enriches a sheep body length molecular marker database, and researches show that the body length of sheep individuals with CC genotypes is obviously higher than that of sheep individuals with genotypes of CA and AA, in the sheep breeding process, CC homozygous individuals with dominant adult body length can be selected and remained, particularly, individuals with dominant body length are selected and remained in the lamb stage, and the SNP molecular marker has potential application value for large-scale molecular breeding of sheep.
On the basis, the invention also provides a primer group and a kit for detecting the SNP molecular marker and a method for detecting and/or detecting sheep body length characters, the primer group or the kit can be used for predicting whether the sheep adult postbody length to be detected has advantages in a lamb stage based on the SNP locus typing, the method has high accuracy, cost performance and sensitivity, the SNP locus can be automatically detected, tens to hundreds of SNP loci in hundreds to thousands of samples can be detected at the same time, and technical support is provided for sheep body length molecular auxiliary breeding.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the results of three genotyping of SNP loci in 382 sheep individuals using Sequenom MassARRAY ® SNP technique in example 1.
Detailed Description
The invention provides an SNP molecular marker related to sheep body length characters, wherein the SNP molecular marker is positioned at 51717733bp locus on chromosome 4 of sheep genome, and the SNP molecular marker has C/A base mutation.
The sheep genome sequence information version number is preferably ARS-UI_ Ramb _v2.0. The nucleotide sequence containing the SNP molecular marker is preferably shown as SEQ ID NO.1 or SEQ ID NO.2, namely, the base at the 201bp position of the nucleotide shown as SEQ ID NO.1 or SEQ ID NO.2 is C or A, specifically, the position of the base underlined by bold italics is the position of the SNP molecular marker.
SEQ ID NO.1：5'-CATCCCACCAGCAAGACTCTTTAGATATCTGAGCAGAGAGATAGCTCTCATTGTCCATGAAACAAGGTCCCTTTCTCTATTCTTTCTGCTAGTAAGTGGGTAGTGATACAATGAGTAATAGGAGAGAGCTGATTCCATCAGCTGGGGTCATAAACACTGAAGTTTCATACACACTTAAGCAACTTGTGACATCCACCCCCCCCCCCCCACCCATCACTGAGGGATGTTTTTGTTTATTGTCCTTCCACAGGTCCCCTAGGATGATCTCTGGGCATGTCTTTTATTCTCTTCAGGCCTGCCTCACCTTCCTTTAAATCTGCTTCCTCCACAGGCAAAGTTGAATCGGTGGTATTGAGTATTCAGTATTTCCTGTATTGGCATCCCTTAGGCACAGACTTAAA-3';
SEQ ID NO.2：5'-CATCCCACCAGCAAGACTCTTTAGATATCTGAGCAGAGAGATAGCTCTCATTGTCCATGAAACAAGGTCCCTTTCTCTATTCTTTCTGCTAGTAAGTGGGTAGTGATACAATGAGTAATAGGAGAGAGCTGATTCCATCAGCTGGGGTCATAAACACTGAAGTTTCATACACACTTAAGCAACTTGTGACATCCACCCCCACCCCCCCACCCATCACTGAGGGATGTTTTTGTTTATTGTCCTTCCACAGGTCCCCTAGGATGATCTCTGGGCATGTCTTTTATTCTCTTCAGGCCTGCCTCACCTTCCTTTAAATCTGCTTCCTCCACAGGCAAAGTTGAATCGGTGGTATTGAGTATTCAGTATTTCCTGTATTGGCATCCCTTAGGCACAGACTTAAA-3'.
The SNP molecular marker has obvious correlation with sheep body length characters, and the body length of individuals with CC genotypes is obviously higher than those of individuals with CA and AA genotypes.
The invention also provides a primer set for detecting the SNP molecular marker in the technical scheme, wherein the primer set comprises an upstream primer, a downstream primer and an extension primer; the nucleotide sequences of the upstream primer, the downstream primer and the extension primer are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
The nucleotide sequences shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are as follows:
SEQ ID NO.3：5'-ACGTTGGATGCCTGTGGAAGGACAATAAAC-3'；
SEQ ID NO.4：5'-ACGTTGGATGTAAGCAACTTGTGACATCC-3'；
SEQ ID NO.5：5'-GTGACATCCACCCCC-3'。
The invention also provides a kit which comprises the primer set and the PCR amplification reagent.
The PCR amplification reagents of the present invention preferably include reagents related to the genotyping of Sequenom MASSARRAY SNP, and further preferably include, but are not limited to dNTPs, taq DNA polymerase, mgCl 2, PCR reaction buffer, shrimp Alkaline Phosphatase (SAP), and iplex reaction reagents. The invention does not limit the dosage of each component in the PCR amplification reagent, and the components can be combined according to the requirement. The use concentration of the upstream primer and the downstream primer is preferably independently 0.45-0.55 mu mol/L, and more preferably independently 0.50 mu mol/L; the concentration of the extension primer in the primer set is preferably 0.6-1.3 mu mol/L. The dNTPs are preferably used at a concentration of 20-30 mu mol/L, more preferably 25 mu mol/L; the use concentration of the Taq DNA polymerase is preferably 4-6U/. Mu.L, more preferably 5U/. Mu.L; the MgCl 2 is preferably used at a concentration of 20-30 mmol/L, more preferably 25mmol/L; the PCR reaction buffer is preferably 10 XPCR reaction buffer; the enzyme activity of the shrimp alkaline phosphatase is preferably 1.7U/MuL. The iplex reagent of the present invention preferably includes iplex Buffer Plus and iplex Terminator, primer mix and iplex enzyme. The kit of the invention preferably further comprises shrimp alkaline phosphatase Buffer. The kit of the invention preferably further comprises standard positive template DNA; the genotype of the standard positive template DNA is preferably CC, and the standard positive template DNA is used as a positive control, so that the accuracy of SNP locus detection is improved.
The invention also provides the SNP molecular marker of the technical scheme, the primer group of the technical scheme or the application of the kit of the technical scheme in sheep body length molecular marker assisted breeding. The sheep body length molecular marker assisted breeding of the invention preferably comprises prediction of sheep adult body length dominance, and more preferably, prediction of whether sheep adult body length traits are dominant or not in the lamb stage. The invention preferably screens sheep with the SNP molecular marker locus of C base for subsequent breeding, more preferably screens homozygous sheep individuals with the SNP locus of CC genotype for subsequent breeding, and retains the advantage of the length and the growth performance of sheep.
Specifically, the invention also provides a method for predicting whether the adult length of sheep has advantages, which comprises the following steps:
Taking genome DNA of sheep to be detected as a template, and performing PCR amplification reaction by using an upstream primer and a downstream primer in the primer set in the technical scheme to obtain a PCR amplification product;
performing digestion reaction on the PCR amplification product to obtain a digested PCR amplification product;
taking the digested PCR amplification product as a template, and performing an extension reaction by using extension primers in the primer set in the technical scheme to obtain an extension product;
genotyping the extension product to obtain a genotype result;
Predicting the adult length of the sheep to be tested according to the genotype result: when the genotype of 51717733bp locus on chromosome 4 of sheep genome is CC genotype, the adult length of sheep to be detected is long, and the sheep has the advantage of body length;
When the genotype of 51717733bp locus on chromosome 4 of sheep genome is CA or AA genotype, the adult length of the sheep to be detected is short, and the sheep to be detected has no advantage of body length.
The invention preferably extracts the genomic DNA of the sheep to be tested. The sheep to be tested according to the invention is preferably a lamb stage sheep. The invention has no special requirements on the types of sheep to be detected, any sheep can be detected by using the method of the invention, and the invention adopts the Hua Mongolian mutton sheep in one embodiment. The method for extracting the genome of the sheep to be detected is not particularly limited, and an animal cell genome extracting method conventional in the art is adopted, in one embodiment of the invention, red blood cells without DNA are removed by utilizing red blood cell lysate to lyse, the red blood cells are released by cell nucleus lysate to release genomic DNA, then protein is removed by selective precipitation of protein precipitation liquid, and finally pure genomic DNA is obtained by isopropanol precipitation and redissolution in DNA dissolution liquid.
After obtaining the genome DNA of the sheep to be detected, the invention uses the genome DNA of the sheep to be detected as a template, and uses the upstream primer and the downstream primer in the primer group to carry out PCR amplification reaction to obtain PCR amplification products. The reaction system of the PCR amplification reaction of the invention is calculated by 5 mu L and preferably comprises the following components: 20-50 ng/. Mu.L of genomic DNA 1 [ mu ] L, PCR reaction buffer 0.5 [ mu ] L, 25mmol/L MgCl 2 0.4 [ mu ] L, 25 [ mu ] mol/L dNTPs 0.1 [ mu ] L, 0.5 [ mu ] mol/L PCR primer mixture 1 [ mu ] L, 5U/[ mu ] L Taq DNA polymerase 0.2 [ mu ] L and deionized water 1.8 [ mu ] L. The procedure for the PCR amplification reaction according to the invention is preferably: pre-denaturation at 95℃for 2min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 60s for 45 cycles; maintained at 72℃for 5min. The PCR amplification product obtained is preferably stored at 4 ℃. And through the PCR amplification reaction, the PCR amplification product contains a DNA fragment where the SNP locus is located.
After the PCR amplification product is obtained, the shrimp alkaline phosphatase is used for carrying out digestion reaction on the PCR amplification product, so that the digested PCR amplification product is obtained. The digestion system of the digestion reaction according to the invention preferably comprises, in terms of 2 μl: shrimp alkaline phosphatase Buffer 0.17 [ mu ] L, shrimp alkaline phosphatase 0.3 [ mu ] L of 1.7U/[ mu ] L and deionized water 1.53 [ mu ] L. The procedure for the digestion reaction according to the invention is preferably: 40min at 37℃and 5min at 85 ℃. The digested PCR amplification product of the present invention is preferably stored at 25 ℃. The digestion reaction of the invention is used for digesting the primer sequence and the rest dNTPs in a PCR amplification reaction system.
After the digested PCR amplification product is obtained, the digested PCR amplification product is used as a template, and the extension primer in the primer group is used for carrying out extension reaction to obtain an extension product. The extension system of the extension reaction according to the invention preferably comprises, in terms of 2 μl: iplex Buffer Plus 0.2.2 [ mu ] L, iplex Terminator 0.2 [ mu ] L, extension primer 0.94 [ mu ] L, iplex enzyme 0.041 [ mu ] L and deionized water 0.619 [ mu ] L.
The procedure for the extension reaction according to the invention is preferably: 94 ℃ for 30s; [94℃for 5s, (52℃for 5s,80℃for 5 s) ], wherein said (52℃for 5s,80℃for 5 s) is performed for 5 cycles, and said [94℃for 5s, (52℃for 5s,80℃for 5 s) ] is performed for 40 cycles; and 3min at 72 ℃. Iplex Buffer Plus, iplex Terminator and iplex enzyme in the extension system according to the invention are preferably derived from the iPLEX ® Gold REAGENT SET kit. In the process of the extension reaction, single base extension is carried out on SNP loci to be detected in an extension system, a locus specific extension primer extends one base at a mutation locus and terminates, and the extension primer is connected with different ddNTPs according to the difference of mutation types to form a molecular weight difference. After the extension product is obtained, the extension product is preferably subjected to resin purification. The method of the present invention for purifying the resin is not particularly limited, and the resin is conventionally used in the art.
After the extension product is obtained, the invention uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to genotype the extension product, and a genotype result is obtained. The invention preferably samples the extension products on a target sheet, detects the molecular weight difference of different extension products by using a mass spectrometer, and analyzes the data to obtain the specific genotype of each mutation site of the sample to be detected, and particularly, in the process of carrying out genotyping by using the matrix-assisted laser desorption ionization time-of-flight mass spectrum, the mass spectrum sample application is preferably carried out by using MASSARRAY NANODISPENSERRS 1000; mass spectrometry is preferably performed using MASSARRAY COMPACT SYSTEM. After mass spectrometry, the invention preferably uses Typer 4.0 software to detect mass spectrum peaks and interprets the genotypes of the target sites of each sample according to the mass spectrum peak diagram.
The method of the invention is based on Sequenom MassARRAY ® SNP technology, and the basic principle of Sequenom MassARRAY ® SNP technology is as follows: firstly amplifying a DNA fragment of a target SNP locus by using an upstream primer and a downstream primer, adding SAP enzyme into an amplified product to digest a primer sequence and residual dNTPs in a reaction system, and then simultaneously carrying out single-base extension on the SNP locus to be detected by using an extension primer, wherein the extension primer with site specificity extends by one base at a mutation locus and is terminated. The extension product will ligate different ddNTPs depending on the difference in mutation type, resulting in a molecular weight difference. After the extension products are purified by resin, the extension products are spotted on a target sheet, the molecular weight difference of different extension products is detected by using a mass spectrometer, and the specific genotypes of the mutation sites can be obtained through data analysis.
After the genotype results are obtained, the invention predicts the adult length of the sheep to be tested according to the genotype results: when the genotype of 51717733bp locus on chromosome 4 of sheep genome is CC genotype, the adult length of sheep to be detected is long, and the sheep has the advantage of body length; when the genotype of 51717733bp locus on chromosome 4 of sheep genome is CA or AA genotype, the adult length of the sheep to be detected is short, and the sheep to be detected has no advantage of body length.
The method is based on Sequenom MassARRAY ® SNP technology, and the sheep with the SNP locus as the CC genotype is screened for subsequent breeding. The method can select genotype CC homozygous individuals with body length advantage, improves sheep production performance, and has great application value for sheep large-scale molecular breeding.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or in accordance with the manufacturer's instructions.
Example 1
Method for detecting genotype of 51717733 locus on chromosome 4 of sheep and predicting whether adult sheep has length advantage by Sequenom MassARRAY ® SNP technology
1. Experimental materials
382 Inner Mongolian meat sheep with adult sheep body length record were selected.
2. Reagent and instrument
Reagent: complete Genotyping REAGENT KIT for Massary Compact 384; gene amplification: ABI GeneAmp 384 Dual;
Mass spectrum sample application: MASSARRAY NANODISPENSERRS1000;
mass spectrometry: MASSARRAY COMPACT SYSTEM;
all reagents and instruments were purchased from beijing junonode biotechnology limited (Beijing Genenode Biotech co., ltd).
3. Extraction of genomic DNA
Jugular vein blood was collected 1mL at 1 month of postnatal age in sheep and anticoagulated with EDTA. Firstly, the red blood cells without DNA are removed by the lysis of the red blood cell lysate, the red blood cells are released by the lysis of the cell nucleus lysate, then the protein precipitation solution is selectively precipitated to remove the protein, and finally, the pure genome DNA is precipitated through isopropanol and redissolved in the DNA lysate.
4. Sequenom MassARRAY ® SNP technique for genotyping
Primer combinations were designed for the 51717733bp site on chromosome 4 in sheep (version number ARS-ui_ Ramb _v2.0 based on sheep genome sequence information):
the nucleotide sequence of the PCR amplification primer is as follows:
Upstream primer F: 5'-ACGTTGGATGCCTGTGGAAGGACAATAAAC-3' (SEQ ID No. 3)
The downstream primer R: 5'-ACGTTGGATGTAAGCAACTTGTGACATCC-3' (SEQ ID No. 4)
The sequences of the extended primers and the extension products are shown in Table 1:
TABLE 1 extended primer sequences and extension products
The above primers were synthesized by the company Juno.
The detection flow is as follows:
1. extracting genome DNA of sheep to be detected; in addition, two standard positive template DNA were added and the genotype at the 51717733bp position on chromosome 4 (version number ARS-UI_ Ramb _v2.0 based on sheep genome sequence information) was CC.
2. Taking genome DNA of sheep to be detected as a template, and carrying out PCR amplification reaction by using an upstream primer F and a downstream primer R;
The reaction system of the PCR amplification reaction was 5. Mu.L: 20-50 ng/mu L of genome DNA 1 mu L, 0.5 mu L of 10 XPCR reaction buffer solution, 0.4 mu L of 25mmol/L MgCl 2, 0.1 mu L of 25 mu mol/L dNTPs, PCR PRIMER mix 1 mu L, 0.2 mu L of 5U/mu LTaq DNA polymerase and 5 mu L of deionized water;
the procedure for the PCR amplification reaction was: 95 ℃ 2min,95 ℃ 30s,56 ℃ 30s,72 ℃ 60s,45 cycles; and at 72℃for 5min.
3. Digesting the PCR amplification product with SAP enzyme; and (3) digesting the PCR amplification product, and mainly removing the residual primer and dNTPs in the reaction product by utilizing SAP enzyme. SAP enzyme digestion system is 2 [ mu ] L: SAP Buffer 0.17 mu L, SAP Enzyme 0.3 mu L, deionized water complement to 2 mu L; the reaction conditions are as follows: storing at 37deg.C for 40min, 85deg.C for 15min, and 25deg.C.
4. Taking the digested PCR amplification product as a template, and performing an extension reaction by using the extension primer S1; the extension reaction system is 2 [ mu ] L: the method comprises the following steps of (1) filling 0.2 mu L of an iplex Buffer, 0.2 mu L of Terminator mix, 0.94 mu L of an extension primer, 0.041 mu L of an iplex Enzyme and supplementing deionized water to 2 mu L;
The extension reaction conditions are as follows: [94℃for 5s, (52℃for 5s,80℃for 5 s) ]; wherein said (52 ℃ 5s,80 ℃ 5 s) is performed for 5 cycles and said [94 ℃ 5s, (52 ℃ 5s,80 ℃ 5 s) ] is performed for 40 cycles.
5. The extension products were analyzed to identify sheep genotypes.
And (3) transferring the extension product after resin purification to a 384-hole SpectroCHIP (Sequenom) chip, performing MALDI-TOF-MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) reaction, detecting a mass spectrum peak by using Typer 4.0.0 software, and judging genotypes of different sample target sites according to a mass spectrum peak diagram.
The mass spectrum of the extension product is shown in FIG. 1, wherein the blue right triangle indicates AA genotype, the yellow inverted triangle indicates TT genotype, and the green square indicates AT genotype.
Statistical results: the statistical results of correlation analysis of the different genotypes at 51717733bp locus on chromosome 4 of the sheep to be tested on adult (2 years old) body length of the Huameng mutton sheep are shown in Table 2.
TABLE 2 correlation analysis of the adult length of Hume mutton sheep with different genotypes at 51717733bp loci on chromosome 4 of sheep to be tested
The lower case letter marked differently in the last column represents a significant difference (P < 0.05).
The results in fig. 1 and table 2 show that: the body length of CC type adult sheep is significantly higher than the other 2 genotypes.
According to the embodiment, the SNP molecular marker related to sheep body length characters has obvious correlation with sheep adult body length characters, and whether sheep adult after adult has body length advantages can be predicted in early stage by typing the SNP locus. The method can realize automatic detection of the SNP locus, and can select CC homozygous individuals with body length advantage in the sheep breeding process, thereby improving the sheep growth performance and having potential application value for sheep large-scale molecular breeding.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (9)
1. The application of the SNP molecular marker in sheep body length molecular marker assisted breeding is characterized in that the SNP molecular marker is positioned at 51717733bp locus on chromosome 4 of sheep genome, and the SNP molecular marker has C/A base mutation; the sheep genome sequence information version number is ARS-UI_ Ramb _v2.0.
2. The application of the primer group for detecting the SNP molecular marker in sheep body length molecular marker assisted breeding is characterized in that the primer group comprises an upstream primer, a downstream primer and an extension primer; the nucleotide sequences of the upstream primer, the downstream primer and the extension primer are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the SNP molecular marker is positioned at 51717733bp locus on chromosome 4 of sheep genome, and the SNP molecular marker has C/A base mutation; the sheep genome sequence information version number is ARS-UI_ Ramb _v2.0.
3. The application of the kit for detecting SNP molecular markers in sheep body length molecular marker assisted breeding is characterized in that the kit comprises a primer group and a PCR amplification reagent; the primer set comprises an upstream primer, a downstream primer and an extension primer; the nucleotide sequences of the upstream primer, the downstream primer and the extension primer are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the SNP molecular marker is positioned at 51717733bp locus on chromosome 4 of sheep genome, and the SNP molecular marker has C/A base mutation; the sheep genome sequence information version number is ARS-UI_ Ramb _v2.0.
4. The use of claim 3, wherein the PCR amplification reagents comprise dNTPs, taq DNA polymerase, mgCl 2, PCR reaction buffer, shrimp alkaline phosphatase, and iplex reaction reagent; the kit also includes standard positive template DNA.
5. The use according to any one of claims 1 to 4, wherein the sheep body length molecular marker assisted breeding comprises prediction of sheep adult body length trait dominance.
6. A method of predicting whether sheep adult length is advantageous comprising the steps of:
taking genome DNA of sheep to be detected as a template, and performing PCR amplification reaction by using an upstream primer and a downstream primer in a primer group to obtain a PCR amplification product;
performing digestion reaction on the PCR amplification product to obtain a digested PCR amplification product;
Taking the digested PCR amplification product as a template, and performing an extension reaction by using extension primers in the primer group to obtain an extension product;
genotyping the extension product to obtain a genotype result;
Predicting the adult length of the sheep to be tested according to the genotype result: when the genotype of 51717733bp locus on chromosome 4 of sheep genome is CC genotype, the adult length of sheep to be detected is long, and the sheep has the advantage of body length;
When the genotype of 51717733bp locus on chromosome 4 of sheep genome is CA or AA genotype, the length of the sheep to be detected after adult is short, and the sheep to be detected does not have the advantage of length;
The primer set comprises an upstream primer, a downstream primer and an extension primer; the nucleotide sequences of the upstream primer, the downstream primer and the extension primer are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
7. The method according to claim 6, wherein the reaction system of the PCR amplification reaction comprises the following components in terms of 5 [ mu ] L: 20-50 ng/mu L of genomic DNA 1 mu L, PCR reaction buffer solution 0.5 mu L, 25mmol/L MgCl 2 0.4 mu L, 25 mu mol/L dNTPs 0.1 mu L, 0.5 mu mol/L PCR primer mixture 1 mu L, 5U/mu L Taq DNA polymerase 0.2 mu L and deionized water 1.8 mu L;
The PCR amplification reaction is performed at 95 ℃ for 2min in a pre-denaturation mode; denaturation at 95℃for 30s, annealing at 56℃for 30s,
Extending at 72 ℃ for 60s, and 45 cycles are total; maintained at 72℃for 5min.
8. The method of claim 6, wherein the digestive system of the digestion reaction comprises, in 2 μl: shrimp alkaline phosphatase Buffer 0.17 [ mu ] L, shrimp alkaline phosphatase 0.3 [ mu ] L of 1.7U/[ mu ] L and deionized water 1.53 [ mu ] L;
The digestion reaction is performed by the following steps: 40min at 37℃and 5min at 85 ℃.
9. The method of claim 6, wherein the extension system of the extension reaction comprises, in 2 μl: iplex Buffer Plus 0.2.2 [ mu ] L, iplex Terminator 0.2 [ mu ] L, extension primer 0.94 [ mu ] L, iplex enzyme 0.041 [ mu ] L and deionized water 0.619 [ mu ] L;
the procedure for the extension reaction comprises 1) and 2) two steps:
1）94℃ 30s；
2) 94℃for 5s, 1 cycle; then, 5 cycles are carried out at 52 ℃ for 5 seconds and 80 ℃ for 5 seconds;
the entire step 2) was carried out for 40 cycles.
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