CN118147322A - Molecular marker related to lambing number in goat ESRRA gene and application thereof - Google Patents
Molecular marker related to lambing number in goat ESRRA gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of goat molecular marker assisted selection, and particularly relates to a molecular marker related to lambing number in a goat ESRRA gene and application thereof. The molecular marker is located at a G.43033775 locus of a 29 # chromosome in a goat genome Capra hircusARS, the C > T base mutation is shown in a nucleotide sequence shown in a sequence table SEQ ID NO.1, the length of the sequence is 1142bp, the mutation locus is located at a 891 # base in the sequence, and the mutation causes Mse I-RFLP polymorphism. The invention also provides a SNP detection method related to the goat lambing number, which comprises related primers and a kit. The invention provides a new method for the auxiliary selection of the goat marks, and the detection method has high detection rate and short time consumption.
Description
Technical Field
The invention belongs to the technical field of goat molecular marker assisted selection, and particularly relates to a molecular marker related to lambing number in a goat ESRRA gene and application thereof.
Background
The mutton has healthy and safe nutrition characteristics, and the proportion of the mutton in meat consumption is continuously increased along with the continuous increase of the requirements of consumers on healthy diet. The most direct method for improving the annual output of mutton is to increase the number of lambs, so that research on how to improve the lamb production performance of goats through breeding has great significance for improving the production and reproduction benefits of goats.
Marker assisted selection (MARKER ASSISTED selection, MAS) is a modern molecular technology that is based on genetic variation and is being developed under the rapid development of animal molecular genetics and modern molecular biotechnology, and is being used to seek ways to increase animal genetics. Currently, there are two molecular marking methods, namely biochemical marking and DNA marking, which are available for genetic research, but biochemical marking has many limitations, so that DNA marking is a popular method in genetic research. DNA molecular markers are genetic markers analyzed at the DNA level, which are used to analyze genetic polymorphisms between species, populations and individuals and to identify molecular markers closely related to economic traits of animals.
Single nucleotide polymorphism (Single nucleotide polymorphism, SNP) refers to mutations of a single nucleotide present in the genome, which can lead to diversity in the nucleotide sequence. The characteristics of Single Nucleotide Polymorphism (SNP) include site diversity, wide distribution, high genetic stability, strong representativeness, easy detection and quick analysis. Therefore, the auxiliary selection of the excellent goat characters by means of SNP is beneficial to the sustainable development of the goat industry in China. PCR-RFLP is a technology capable of genotyping polymorphic sites, and its principle is that when amplifying identical DNA sequences of different individuals, the restriction enzyme recognition sites are changed due to base mutation, the polymorphic DNA sequences can generate different DNA sequence fragments after the action of the restriction enzyme, and the different size strips can be separated by electrophoresis, so that the identification of each genotype can be completed.
Estrogen-related receptor A (Estrogen-relAtedreceptorsA, ESRRA or ESRR. Alpha.) was Giguere Vincentl, etc. screened from a human heart and kidney cDNA library. mESRRA are expressed primarily in tissues of the body involved in lipid metabolism and energy balance. So far, no related research report on goat ESRRA gene as a molecular marker of goat lambing number character exists.
Disclosure of Invention
The invention aims to provide an application of a molecular marker in a goat ESRRA gene in detecting the lambing number character of goats, which can assist in breeding the lambing number character of goats.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides an application of a molecular marker in a goat ESRRA gene in detecting the lambing number character of the goat, wherein the molecular marker is C & gtT basic group mutation at g.43033775 locus in a goat genome Capra hircusARS 1.
The invention provides an application of a molecular marker in a goat ESRRA gene in detecting the lambing number character of the goat, wherein the molecular marker is the 891 th C & gtT base mutation at the 5' end of a sequence shown as SEQ ID NO. 1 in a goat genome
The invention provides a method for detecting the lambing number character of goats, which comprises the step of detecting C & gtT base mutation at g.43033775 locus in goat genome Capra hircusARS.
The invention provides a method for detecting the lambing number character of goats, which comprises the step of detecting 891 th C & gtT base mutation at the 5' end of a sequence shown as SEQ ID NO.1 in a goat genome.
Preferably, the method is to carry out PCR amplification by using a primer pair, and the sequence of the primer pair is shown as SEQ ID NO. 2-3.
Preferably, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 98 ℃ for 45s; denaturation at 98℃for 10s; the annealing temperature is set to 58 ℃ and the duration is 30s; extending at 72 ℃ for 27s; the cycle number of denaturation, annealing and extension is 34 times; finally, the extension is carried out for 5min at 72 ℃.
Preferably, the above method further comprises an enzyme cleavage type in which the nucleotide sequence shown in SEQ ID NO. 1 is subjected to an enzyme cleavage treatment with restriction enzyme Mse I.
Preferably, the product after cleavage is detected by 2% agarose gel electrophoresis.
The invention also provides a kit for detecting the lambing number of goats, which comprises primer pairs shown as SEQ ID NO. 2-3.
Preferably, the goat is Chu Bao black-head sheep.
The invention has the beneficial effects of.
The invention provides a molecular marker related to the lambing number in a goat ESRRA gene, and by checking the molecular marker, the female sheep individuals with more lambing numbers can be screened, so that the breeding efficiency is improved. In addition, the method for detecting the goat lambing number character has the advantages of high detection rate, short time consumption and the like, and is convenient for industrial application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the result of gel electrophoresis of goat ESRRA gene fragment SEQ ID NO. 1.
FIG. 2 is a graph showing the detection results of Mse I-RFLP, in which lanes: lanes M are DL2000 Marker, lane 3 is CC type, lanes 1, 4, 5 and 6 are CT type, and lanes 8 and 9 are TT type.
Detailed Description
The invention provides an application of a molecular marker in a goat ESRRA gene in detecting the lambing number character of the goat, wherein the molecular marker is C & gtT base mutation at g.43033775 locus in Capra hircusARS of genome sequence information version number. The molecular marker is also the mutation of 891 th C & gtT base at the 5' end of the sequence shown in SEQ ID NO. 1 in goat genome. The mutation is an allelic mutation, preferably, the number of lambing of CT genotype ewe individuals is the greatest.
The invention also provides a detection method of the label, a detection primer and a kit. The detection primer is preferably a primer pair shown as SEQ ID NO. 2-3.
The invention does not limit the mode of detecting the mutation of 891 th C & gtT base at the 5' end of the sequence shown as SEQ ID NO. 1 in the goat genome, and can be a mode of gene sequencing and the like, preferably an enzyme cutting mode. The enzyme used for the cleavage type is the restriction enzyme Mse I.
The checking method utilizes a PCR amplification method to amplify a target sequence, and the PCR amplification reaction program is as follows: pre-denaturation at 98 ℃ for 45s; denaturation at 98℃for 10s; the annealing temperature is set to 58 ℃ and the duration is 30s; extending at 72 ℃ for 27s; the cycle number of denaturation, annealing and extension is 34 times; finally, the extension is carried out for 5min at 72 ℃.
The goat in the present invention is preferably Chu Bao black bellwether.
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 production process, the experimental method or the detection method related to the embodiment of the invention are all conventional methods in the prior art unless otherwise specified, and the names and/or the abbreviations thereof are all conventional names in the field, so that the related application fields are very clear and definite, and a person skilled in the art can understand the conventional process steps according to the names and apply corresponding equipment to implement according to conventional conditions or conditions suggested by manufacturers.
The various instruments, equipment, materials or reagents used in the examples of the present invention are not particularly limited in source, and may be conventional products commercially available through regular commercial routes or may be prepared according to conventional methods well known to those skilled in the art.
Example 1
Method for obtaining goat ESRRA gene fragment (SEQ ID NO: 1) and detecting polymorphic site
1. Extraction of goat genomic DNA
The Chu Bao black-head sheep, which is a mutton sheep improved by Macheng black goats, is selected as a test animal, and the sample is derived from a sheep farm of the national academy of agricultural sciences, livestock and veterinary research institute, hubei province. The whole genome DNA of the goat is extracted by adopting a blood genome DNA extraction kit (produced by Beijing Tiangen Biochemical technology Co., ltd.) and the specific steps are referred to the kit instruction. And (3) detecting the concentration and quality of the obtained genome DNA, marking the number, and storing in a refrigerator at the temperature of minus 80 ℃ for standby.
2. Acquisition of SNP genetic marker detection fragment
(1) PCR amplification
Primers were designed based on goat ESRRA genomic sequence (Gene ID: 102175649) and the primer sequence information was as follows:
upstream primer (SEQ ID NO: 2): CGTCTTCCCTTCATCTGTTCG (5 '. Fwdarw.3') downstream primer (SEQ ID NO: 3): TACTCGCTGCTCCTGCCCAT (5 '. Fwdarw.3')
PCR amplification was performed using the above primer pair Chu Bao black bellwether genomic DNA. The reaction system is shown in Table 1.
TABLE 1PCR reaction System
The PCR reaction program was set as follows: pre-denaturation at 98 ℃ for 45s; denaturation at 98℃for 10s; the annealing temperature is set to 58 ℃ and the duration is 30s; extending at 72 ℃ for 27s; the cycle number is fixed, 34×; final extension at 72℃for 5min;12℃for 1min. The PCR amplification product was stored at 4 ℃.
The amplified product is goat ESRRA gene fragment, the sequence of which is shown as SEQ ID NO. 1, total 1142bp, and the result of gel electrophoresis is shown as figure 1.
The 891 base in the sequence has C/T allele mutation, which causes the restriction enzyme Mse I cleavage site to change through NEB website online detection, and when the base is T, the cleavage site appears. This site is on intron 6 of the goat ESRRA gene.
Method for detecting mutant type by means of enzyme cutting type (Mse I-RFLP detection)
10. Mu.L of the PCR product was taken in a PCR tube, 0.2. Mu.L of restriction enzyme Mse I, 1. Mu.L of 10 Xbuffer and 3.8. Mu. LddH2O were added thereto, and the digested product was subjected to digestion (1 to 16) for 1 to 16 hours at 37℃and detected by 2% agarose gel electrophoresis, and the digestion result was observed and recorded under a gel imaging system. When the base at the position is C (i.e. when no mutation occurs), the enzyme cutting site completely disappears, and only one band (1142 bp) is observed after enzyme cutting, and the CC genotype is marked; when both the C and T bases at this position exist (i.e., they are shown as heterozygous mutations), the result of the cleavage is three bands (250 bp+891bp+1142bp), designated as CT genotype; when the bases at this position were all T (i.e., homozygous mutation occurred), two bands (250 bp+891bp) were observed after cleavage and were noted as TT genotypes, and the results are shown in FIG. 2.
Example 2
Polymorphism distribution detection of molecular markers in goat populations
In this example, polymorphism detection was performed by detecting the g.430337754C > T site on the ESRRA gene of goat in Chu Bao black bellwether population, and the detection results are shown in Table 2.
TABLE 2 genotype frequencies and Gene frequencies of goat ESRRA Gene g.43033775C > T locus
From the results in Table 2, it can be seen that: goat ESRRA gene g.43033775c > T site there are three genotype individuals in Chu Bao black bellwether population, namely CC, CT, TT genotypes, with homozygous CC type as the dominant genotype and allele C with a genetic frequency of 0.69. Through chi-square test, the genotype distribution of the locus does not accord with the Hardy-Winberg equilibrium state.
Example 3
Correlation verification of molecular marker and goat lambing character of the invention
500 Black goats Chu Bao are selected as test materials, and the sample collection and the related lambing information are all from the sheep farm of the national academy of agricultural sciences of Hubei province. Polymorphism examination was performed using PCR-BmgB I-RFLP, and correlation between individuals of the corresponding different genotypes and their lambing traits was analyzed. Performing association analysis between individual traits of different genotypes of molecular markers by adopting a GLM program in SAS statistical analysis software, wherein the model is as follows:
model 1: y=global mean + genotype + sheep field environmental effect + residual
Model 2: y=overall mean + additive effect + dominant effect + sheep field environmental effect + residual.
Wherein Y is a character phenotype value. Additive effect= (homozygote 1-homozygote 2)/2, homozygote 1, homozygote 2 are represented by 1,0, -1, respectively; dominant effect = heterozygote- (homozygote1 + homozygote2)/2, homozygote 1, homozygote 2 being represented by 1, -1, respectively. The statistical analysis results are shown in table 3:
TABLE 3 analysis of the correlation of ESRRA Gene g.39080534G > A with lambing number
Note that: the first column of the table indicates the total lamb nest count for the same parity. The values are least square values plus or minus standard error. In the same line comparison, the different lower case letters represent significant differences (P < 0.05)
As can be seen from table 3, in Chu Bao black bellwether population, the CT heterozygous mutant individuals were 0.2 head higher (P < 0.05) than the CC individuals for the number of calves; for average lambing numbers, CT heterozygous mutant individuals were 0.14 head higher (P < 0.05) than CC type individuals. Thus, g.43033775C > T locus CT genotype ewe individuals had more lambing numbers.
According to the embodiment, the molecular marker in the goat ESRRA gene can be applied to detecting the character of the lambing number of the goat, so that the screening of the ewe with high lambing number is facilitated, the application detection rate is high, the time consumption is short, and the industrial application is facilitated.
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 (10)
1. An application of a molecular marker in a goat ESRRA gene in detecting the lambing number character of the goat, wherein the molecular marker is C & gtT base mutation at g.43033775 locus in goat genome Capra hircus ARS 1.
2. The application of a molecular marker in a goat ESRRA gene in detecting the lambing number character of the goat is that the 891 th C & gtT base mutation of the 5' end of a sequence shown as SEQ ID NO. 1 in a goat genome.
3. A method for detecting a lambing number trait in a goat, the method comprising detecting a C > T base mutation at the g.43033775 locus in the genome Capra hircusARS of the goat.
4. A method for detecting the lambing number character of goats is characterized by comprising the step of detecting 891 th C & gtT base mutation at the 5' end of a sequence shown as SEQ ID NO. 1 in a goat genome.
5. The method of claim 4, wherein the method comprises PCR amplification using a primer pair having the sequence set forth in SEQ ID NO. 2-3.
6. The method of claim 5, wherein the PCR amplification reaction procedure is: pre-denaturation at 98 ℃ for 45s; denaturation at 98℃for 10s; the annealing temperature is set to 58 ℃ and the duration is 30s; extending at 72 ℃ for 27s; the cycle number of denaturation, annealing and extension is 34 times; finally, the extension is carried out for 5min at 72 ℃.
7. The method according to claim 4, wherein the method comprises an enzyme cleavage type in which the nucleotide sequence shown in SEQ ID NO. 1 is subjected to an enzyme cleavage treatment with restriction enzyme MseI.
8. The method of claim 7, wherein the digested product is detected by 2% agarose gel electrophoresis.
9. A kit for detecting the lambing number of goats is characterized by comprising primer pairs shown as SEQ ID NO. 2-3.
10. The use, method or kit according to any one of claims 1 to 9, wherein the goat is Chu Bao black-head sheep.
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