CN118147321A - Application of molecular marker g.390806766G > A in PRLR gene in detection of goat lambing number traits - Google Patents
Application of molecular marker g.390806766G > A in PRLR gene in detection of goat lambing number traits Download PDFInfo
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Abstract
The invention belongs to the technical field of auxiliary selection of goat molecular markers, and particularly relates to application of a molecular marker g.390806766G > A in a PRLR gene in detecting goat lambing number traits. The molecular marker can be used for detecting the lambing character of goats, screening goats with more lambing numbers, and greatly improving the breeding efficiency. The results of the examples show that for average lambing numbers, GA heterozygous mutant individuals are 0.34, 0.64 head (P < 0.05) higher than GG, AA-type individuals; thus, g.39080676G > A locus GA genotype ewe individuals were able to obtain more lambing numbers.
Description
Technical Field
The invention belongs to the technical field of auxiliary selection of goat molecular markers, and particularly relates to application of a molecular marker g.390806766G > A in a PRLR gene in detecting goat lambing number traits.
Background
The mutton has healthy and safe nutrition characteristics, and the goat feeding history in China is long, so that the mutton is a common resident meat source. With the increasing demand of consumers for healthy diet, the specific gravity of mutton in meat consumption is continuously increased. 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.
Prolactin receptor (PRLR) is a specific receptor for Prolactin (PRL), a member of the prolactin receptor superfamily, and by binding to PRL, modulates reproductive performance in females, and mutations in this gene affect the age of females to primordia. PRLR genes were first found in rats and then in mice, and scholars cloned the PRLR genes in other animals such as monkeys, cows, horses, pigs, chickens, geese, etc. Researchers at home and abroad develop a great deal of research on the influence of PRLR genes on lactation and reproduction traits of livestock and poultry. For example, the SNP mutation at one side wing region of the PRLR gene is obviously related to the average lactation yield and the milk protein of ARUM Yue Lv, and can be used as an effective DNA marker for breeding of the milk-type ARUM donkey. In addition, the PRLR gene can also be used as an important candidate gene for lamb litter size and sea goats breeding. To date, there has been no report on the study of the goat PRLR gene as a molecular marker for Chu Bao black bellwether lambing trait.
Disclosure of Invention
The invention aims to provide an application of a molecular marker in a PRLR gene in detecting the goat lambing number character, and the application can assist in breeding the goat lambing number character.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides application of a molecular marker in a PRLR gene in detecting goat lambing number characters, wherein the molecular marker is G > A base mutation at g.39080676 in goat genome Capra hircusARS.
The invention also provides application of the molecular marker in the goat PRLR gene in detecting the lambing number character of the goat, wherein the molecular marker is the 608 th G > A base mutation of the 5' end of the sequence shown as SEQ ID NO. 1 in the goat genome.
The invention provides a primer pair for detecting the molecular marker, and the sequence of the primer pair is shown as SEQ ID NO. 2-3.
The invention provides a method for detecting the lambing number character of goats, which comprises the steps of detecting G > A base mutation at g.39080676 locus in goats genome Capra hircusARS or detecting G > A base mutation at 608 th locus of 5' end of a sequence shown as SEQ ID NO. 1 in goats genome.
Preferably, the above detection method comprises the steps of amplifying a nucleotide sequence containing the above molecular marker locus to obtain an amplified product, and then performing an enzyme cleavage type in which the amplified product is subjected to an enzyme cleavage treatment with a restriction enzyme MIuc I.
Preferably, the method for amplifying the molecular marker loci comprises the steps of carrying out PCR amplification by using a primer pair, wherein the sequence of the primer pair is shown as SEQ ID NO. 2-3, 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 55 ℃ and the duration is 30s; extending at 72 ℃ for 25s; the cycle number of denaturation, annealing and extension is 34 times; finally, the extension is carried out for 5min at 72 ℃.
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 beneficial effects are that:
The molecular marker in the PRLR gene of the goat can be used for detecting the character of the lambing number of the goat, assisting in breeding the goat with a large lambing number, and can obviously improve the breeding efficiency. In addition, the method for detecting the goat lambing number character has high detection rate and short time consumption, 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 results of gel electrophoresis of Chu Bao black bellwether PRLR gene fragment SEQ ID NO: 1.
FIG. 2 is a diagram showing the results of the RFLP assay of example 1MIuc I, in which lanes M are DL2000Marker, lanes 3 are GG type, lanes 1, 4, 5, 6 are GA type, and lanes 8, 9 are AA type.
Detailed Description
The invention provides an application of a molecular marker g.390806766G > A in a PRLR gene in detecting goat lambing number traits, wherein the molecular marker is G > A base mutation at g.39080676 in a Capra hircus ARS1 with a genome sequence information version number. The molecular marker is also the mutation of the base G > A at the 608 th position of the 5' end of the sequence shown as SEQ ID NO. 1 in the goat genome. The mutation is an allelic mutation, preferably, the GA genotype ewe individuals have the greatest lambing number.
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 the 608 th G > A base at the 5' end of the sequence shown in 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 is restriction enzyme MIucI.
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 55 ℃ and the duration is 30s; extending at 72 ℃ for 25s; 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 may be any sheep species comprising the molecular markers of the present invention, 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
Acquisition of goat PRLR gene SNP detection fragment (SEQ ID NO: 1) and polymorphic site detection method
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 the goat PRLR genomic sequence (Gene ID: 102175649), and the primer sequence information was as follows:
Upstream primer (SEQ ID NO: 2): TAATAGAAGGGCAAGTCCGA (5 '. Fwdarw.3')
Downstream primer (SEQ ID NO: 3): CAACCCATCCTTCCATAACTC (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 be 52 ℃ and the duration is 30s; extending at 72 ℃ for 25s; 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 PRLR gene fragment, the sequence of which is shown as SEQ ID NO.1, and the total of 1025bp, and the result of gel electrophoresis is shown as figure 1.
The 608 th base in the sequence has G/A base mutation, which causes the restriction enzyme MIucI to change, and the A mutation causes MIuc I to change, when the base is A, the restriction enzyme is detected on line by NEB website. This site is on exon 9 of the goat ESRRA gene.
Polymorphism site detection method MIucI-RFLP detection
10. Mu.L of the PCR product was taken in a PCR tube, 0.2. Mu.L of restriction enzyme MIucI, 1. Mu.L of 10 Xbuffer and 3.8. Mu. LddH 2 O 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 G (i.e. when no mutation occurs), the enzyme cutting site completely disappears, and only one band (1025 bp) is observed after enzyme cutting, and the GG genotype is marked; when the base at the position G and the base at the position A exist (namely, the base shows heterozygous mutation), the enzyme digestion result is three bands (417 bp+608bp+1025 bp) which are marked as GA genotype; when the bases at this position were all A (i.e., homozygous mutation occurred), two bands (417 bp+608 bp) were observed after cleavage and were designated as AA genotype, and the results are shown in FIG. 2.
Example 2
Polymorphism distribution detection of the molecular marker in goat population
In this example, polymorphism detection was performed by detecting the g.390806766G > A site on the goat PRLR gene in Chu Bao black bellwether population, and the detection results are shown in Table 2.
TABLE 2 genotype frequencies and Gene frequencies of goat PRLR Gene g.390806766G > A locus
From the results in Table 2, it can be seen that: the goat PRLR gene g.390806766G > a locus there are three genotype individuals in Chu Bao black bellwether population, namely GG, GA, AA genotypes, with homozygous GG type as the dominant genotype and allele G gene frequency of 0.95. 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-MIucI-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 with 1, 0,
-1 Represents homozygote 1, heterozygote, homozygote 2, 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 PRLR Gene g.390806767G > A and lambing number correlation
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 row comparison, the different lower case letters represent significant differences (P < 0.05).
As can be seen from Table 3, in the Chu Bao black bellwether population, the GA heterozygous mutant individuals were 0.48, 0.84 head (P < 0.05) higher than the GG, AA type individuals for the number of calves; for the number of lambings on birth 4, the GA heterozygous mutant individuals were 1.07 head higher (P < 0.05) than the AA homozygous mutant individuals; for the number of 5 fetuses, the GA heterozygous mutant individuals were 1.05 head higher (P < 0.05) than the AA homozygous mutant individuals; for average lambing numbers, GA heterozygous mutant individuals were 0.34, 0.64 head higher (P < 0.05) than GG, AA type individuals; for the number of meristematic lambs, the GA heterozygous mutant individuals were 0.39 head higher (P < 0.05) than the AA homozygous mutant individuals. Thus, g.39080676G > A locus GA genotype ewe individuals were able to obtain more lambing numbers.
From the above examples, it is known that the molecular marker g.390806766G > A in the PRLR gene provided by the invention can be applied to detecting the lambing number character of goats.
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)
- The use of a molecular marker in the prlr gene for detecting the lambing number trait in goats, wherein the molecular marker is a G > a base mutation at g.39080676 in the genome Capra hircusARS of goats.
- 2. The application of the molecular marker in the PRLR gene of the goat in detecting the lambing number character of the goat is characterized in that the molecular marker is the 5' end 608 th G > A base mutation of the sequence shown as SEQ ID NO. 1 in the genome of the goat.
- 3. A primer pair for detecting the molecular marker according to claim 1 or 2, wherein the sequence of the primer pair is shown in SEQ ID No. 2-3.
- 4. A method for detecting a goat lambing number trait comprising detecting a molecular marker according to claim 1 or 2.
- 5. The method according to claim 4, wherein the detection method comprises the steps of: amplifying the nucleotide sequence containing the molecular marker loci to obtain an amplified product, and then performing enzyme digestion type, wherein the enzyme digestion type is to perform enzyme digestion treatment on the amplified product by using restriction enzyme MIuc I.
- 6. The method of claim 5, wherein the method of amplifying the molecular marker loci comprises PCR amplification using a primer pair having the sequence shown in SEQ ID NO. 2-3, wherein the PCR amplification is performed by the following reaction procedure: pre-denaturation at 98 ℃ for 45s; denaturation at 98℃for 10s; the annealing temperature is set to 55 ℃ and the duration is 30s; extending at 72 ℃ for 25s; the cycle number of denaturation, annealing and extension is 34 times; finally, the extension is carried out for 5min at 72 ℃.
- 7. The method of claim 5, wherein the detection method further comprises detecting the digested product by 2% agarose gel electrophoresis.
- 8. A kit for detecting the lambing number character of goats is characterized by comprising primer pairs shown as SEQ ID NO. 2-3.
- 9. The use according to any one of claims 1-2, or the kit according to claim 8, wherein the goat is Chu Bao black-head sheep.
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