CN114717332A - SNP molecular marker related to poultry growth and meat quality traits and application thereof - Google Patents
SNP molecular marker related to poultry growth and meat quality traits and application thereof Download PDFInfo
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Abstract
The invention provides an SNP molecular marker related to poultry growth and meat quality traits and application thereof. The SNP molecular marker is positioned in the tenth exon sequence of the GHR gene, the mutation site is A1514T, and the genotype of the SNP site is AA, AT and TT. The SNP molecular marker is obviously related to a plurality of traits of poultry such as growth, carcass, meat quality and the like, and is a novel molecular marker. The growth traits and carcass traits of AT genotype individuals such as living body weight, breast and leg muscle weight, wing weight, head and foot weight, heart and liver muscle, stomach and gland weight, small intestine length and the like are obviously higher than those of AA and TT genotype individuals, and the subcutaneous fat thickness of the AA genotype individuals is obviously higher than that of the AT and TT genotype individuals. The meat quality characters such as the chest muscle shearing force, the leg muscle conductivity and the like of the TT genotype individual are obviously lower than those of the AA and AT genotype individuals. The growth and meat quality traits of the poultry are selected early by determining the genotype of the SNP locus, so that the genetic progress of the weight and the meat quality traits of the poultry can be accelerated, the production performance of the poultry is improved, the production cost is saved, and the economic benefit of the poultry is improved.
Description
Technical Field
The invention belongs to the technical field of molecular biotechnology and molecular markers, and particularly relates to an SNP molecular marker related to poultry growth and meat quality traits and application thereof.
Background
On the growth axis of poultry, GH gene is an important hormone for regulating animal growth, and has the function of promoting growth. When GH functions, the first step is to bind to GH receptors on the surface of target cells and then to signal into the cell via GHR, promoting the activity of pathways associated with intracellular growth. Growth Hormone Receptors (GHRs) are key genes for GH to regulate cell growth. Growth Hormone Receptor (GHR) genes have also been reported to be associated with fat deposition in chickens. During growth and development of animals, growth hormone must bind to growth hormone receptors in order for growth hormone to function. In the case of cattle, when the GHR gene base sequence is mutated, the normal function of GH is affected, and further, various properties such as milk production and meat production are affected. Research shows that the growth performance of the broiler chicken can be improved when the secretion of the growth hormone is increased, the metabolism of animal substances and energy is promoted, and the growth and development processes are promoted. Meanwhile, the GH gene is also an important functional gene for poultry and plays a key role in the growth and development of the poultry.
GHR is the basis of the normal function of GH and has close relation with the growth and development of animals. GHR is an important transmembrane glycoprotein, is encoded by a single gene, contains 620 amino acids, and can normally play the roles of prolactin, cytokines, growth hormone, erythropoietin and the like when organs in an animal body contain enough GHR. GHR exists in most cells of the body, plays a role in combining with GH to play a physiological role of GH, and is particularly prominent in the liver, and the liver is an organ which most expresses chicken GHR in the research of chicken GHR. In addition to the liver, the GHR gene is expressed in skin, heart, muscle, lung, kidney, testis, ovary, adrenal gland, brain, and lymphoid tissue, etc. However, different locations of the GHR gene can also function differently in different organs, with different regulation methods. Researches show that the expression of the GH gene can affect the birth weight, the weaning weight and the 1-year weight of the cattle. The transmembrane structure of the chicken GHR receptor is positioned at 24 amino acid residues of 238 th-261 st channel, the GHR gene contains 592 amino acid residues, and a 16 amino acid signal peptide is found at the N terminal. The sequence of chicken GHR amino acid residues has a relatively low homology to mammals, for example, 53% in chicken and rabbit and 58% in mouse. On the contrary, the GHR homology is higher among mammals, for example, the homology between human and mouse and rabbit reaches 70% and 84%, respectively, and although the homology is not high, the GHR structures are similar and all consist of three parts, namely extracellular structure, intracellular structure and transmembrane structure. Wherein the extracellular structure is responsible for binding to the extramembranous ligand, and the disulfide bond formed by 7 cysteines present in the extracellular structure enables GHR to maintain a specific spatial structure extracellularly without deformation.
With the continuous development of molecular marker technology and molecular quantitative genetics, the generation interval of breeding can be greatly shortened and the genetic progress of breeding can be accelerated by applying molecular genetic markers to carry out marker-assisted selection. Single Nucleotide Polymorphisms (SNPs) mainly refer to DNA sequence Polymorphisms caused by variation of a Single Nucleotide at the genome level, have the characteristics of easy statistics, wide distribution, good genetic stability and the like, are genetic marker technologies most widely applied at present, and play an important role in the field of animal genetic breeding research. Local varieties in China are rich in resources, and most local varieties of poultry have excellent properties such as strong stress resistance, weak nesting property and the like. With the improvement of living standard and the change of diet concept, people have higher requirements on the meat quality of poultry. Local varieties are popular with consumers due to good meat quality and unique flavor, and have large market demand, but in recent decades, the breeding industry has paid excessive attention to the weight and growth speed of poultry, so that excessive fat deposition in poultry bodies is caused, and the meat quality traits of the poultry are influenced. The GHR gene of the poultry is positioned on a chromosome 5, has the gene length of 87kb, comprises 10 exons, searches SNPs related to the growth and meat quality traits of the poultry, is applied to molecular breeding, can accelerate genetic progress and save production cost, and provides valuable information for poultry breeding.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide an SNP molecular marker related to poultry growth and meat quality traits, establish a marker genotype detection method, apply the marker genotype detection method to poultry breeding and improve poultry carcass traits.
The first aspect of the invention provides an SNP molecular marker related to poultry growth and meat quality traits, the SNP molecular marker is positioned in a galGal6 version genome Z chromosome, a mutation site in a tenth exon sequence corresponding to a GHR gene is A1514T, and the genotype of the SNP site is AA, AT and TT.
In a second aspect of the invention, there is provided a method for early selection in connection with poultry growth and meat quality traits. Early selection of poultry is carried out according to the genotype of the mutation site A1514T in the tenth exon region of the GHR gene of the poultry to be detected, individuals with expected carcass traits meeting expected targets are obtained for feeding, the overall feeding cost is reduced, and the output value is improved.
Further, the early selection method comprises the following steps:
1) extracting DNA of poultry blood to be detected;
2) carrying out PCR amplification by taking the blood DNA of the poultry to be detected as a template to obtain a PCR product containing a GHR gene target fragment;
3) detecting the genotype of a mutation site A1514T of the PCR product by adopting a DNA sequencing method;
4) and (3) early selecting the growth and meat quality traits of the poultry based on the genotype of the SNP locus in the step 3), wherein the growth and carcass traits such as the body weight and the like of the AT genotype individual are obviously stronger than those of the AA and TT genotype individuals, and the meat quality traits of the TT genotype individual are obviously lower than those of the AA and AT genotype individuals. During breeding, individuals with AT genotypes are selected, so that the growth and meat quality traits of the poultry are improved.
Further, the nucleotide sequence of the primer used for the PCR amplification in the step 3) is as follows:
the upstream primer PCR-F: 5'-CCCTGACAAACACTGAC-3' (SEQ ID NO. 1);
downstream primer PCR-R: 5'-ACACCCACAAGAACAAG-3' (SEQ ID NO. 2).
Compared with the prior art, the invention has the following beneficial effects:
the SNP molecular marker provided by the invention, namely the mutation site A1514T in the exon 10 sequence of the GHR gene of poultry is related to the growth and meat quality traits of poultry, and is a novel molecular marker. The growth and meat quality traits of the poultry can be improved by determining the genotype of the SNP locus for early selection, the production cost is saved, the genetic progress is accelerated, the SNP locus is better applied to poultry breeding, and the SNP locus has great economic application value and scientific research value.
Drawings
FIG. 1 is a graph showing the genotyping results of mutation site A1514T in the GHR gene.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 extraction of blood DNA to be tested from the Pleurotus eryngii var. sinica (L.) Nees F2 generation of resource group holomorphic line
The DNA sample is a full sibling F2 resource population DNA sample of apricot blossom chicken and recessive white rock chicken stored in the laboratory. Recessive white rook chicken is a fast large broiler, while apricot blossom chicken is a local variety of broiler in China. The second generation broiler chickens hybridized by the apricot blossom chickens and the recessive white rooks are fed with the corn soybean feed meeting the international formula standard in a flat-feeding mode. Slaughter samples were taken at 90 days of age and their shin length, head width, chest depth, body length, chest angle width, slaughter weight, cortical thickness, evisceration weight, bore weight, pectoral weight, leg weight, feather weight, abdominal fat weight, head and neck weight, heart weight, liver weight, stomach weight and small intestine length were recorded. Wherein the shin length, head width, chest depth and body length are measured by a vernier caliper; measuring the chest angle by a chest angle meter; subcutaneous fat thickness was measured at the back near the tail, fat width was measured between the leg and pectoral muscles, both of which were measured with a vernier caliper.
Genomic DNA of all individuals
The DNA from blood was extracted by the Plant Mini Kit (Qiagen, Hilden, CA; Cat #69104) Kit instructions, diluted to 50 ng/. mu.L after quality and concentration testing, and stored at 4 ℃ until use.
Example 2 SNP detection and Mixed pool DNA sequencing
1. Designing primers and detecting specificity:
based on GHR gene sequences published in GeneBank, primer sequences for amplifying the GHR gene were designed as shown in Table 1. The designed primer pair sequence is synthesized by the biological company of biology (Shanghai), and the primer pair is used for carrying out PCR amplification on the GHR gene standard substance to test the specificity of the primer. The nucleotide sequence of the PCR amplification product of the GHR gene standard product is shown as SED ID NO.3, and the SNP site is positioned at the 116 th base of the amplified 689bp fragment sequence.
TABLE 1 primer information for SNP screening of GHR
2. DNA mixed pool sequencing:
randomly selecting 10 DNA samples from the total extracted DNA samples to construct a mixed pool, carrying out PCR amplification on the mixed pool samples, wherein the used primers are the same as the primer pairs used in the SNP detection, and the reaction system is shown in Table 2.
TABLE 2 PCR reaction System
PCR reaction procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 1min, and 37 cycles; extending for 10min after 72 ℃; storing at 4 ℃.
And (3) sending the obtained PCR product to a biological company for sequencing, and detecting the mutation site A1514T in the tenth exon sequence of the GHR gene of the poultry according to the sequencing result, wherein the analysis of the sequencing result is shown in the attached figure 1.
Example 3 genotyping of GHR mutation site A1514T
All of the blood DNA obtained in example 1 was subjected to PCR-specific amplification using the primer set of step 1 in example 2, to obtain a PCR product of a target fragment of the GHR gene. The PCR products were sequenced separately, and the genotype of each sequencing result was analyzed, with the typing results shown in FIG. 1. 199 individuals were successfully typed in this example, and then each genotype was statistically analyzed to obtain the genotype frequency and allele frequency at that site. The gene and genotype frequency statistical structure of the SNP mutation site A1514T is shown in the following table 2.
TABLE 3 genotype frequency allele frequency of GHR Gene mutation site A1514T
Wherein:
(1) genotype frequency refers to the ratio of the number of individuals of a certain genotype in a population to the total number of genotypes:
genotype frequency ═ the total number of certain genotypes/the total number of the population × 100%;
(2) the gene frequency refers to the ratio of a certain gene in a population to all genes at the same site:
the gene frequency is equal to the number of certain genes/the total number of genes at the same site in a population multiplied by 100%.
As can be seen from the structure in Table 3, the AA genotype is the dominant genotype of the chicken population to be tested.
Example 4 Association analysis of SNP mutation site A1514T with poultry growth and meat quality traits
The results of the association analysis of the different genotypes of the GHR gene with the growth and carcass traits of this F2 population were expressed as (mean ± standard error) using SPSS software. Significance analysis was performed by t-test, P <0.05 indicated significant difference, and P <0.01 indicated very significant difference. The correlation analysis results are shown in table 4 below.
TABLE 4 GHR Gene mutation site A1514T correlation analysis with poultry growth, carcass and meat quality traits
Note: the same letter in the same row indicates no significant association and a different letter indicates significant association (P < 0.05).
From the table, it can be found that the mutation site is significantly related to multiple traits of poultry, such as live weight, breast weight, leg weight, wing weight, head and neck weight, foot weight, heart liver muscle, stomach and gland weight, small intestine length, carcass weight, chest angle, full bore weight, half bore weight, chest muscle shear force, leg muscle conductivity, body weight 28, body weight 35, body weight 42, shin length 42, shin diameter 42, body weight 49, shin length 49, shin diameter 49, etc. (P < 0.05). Wherein, the growth and carcass traits such as the body weight of the AT genotype individual are obviously stronger than those of the AA and TT genotype individuals, and the meat quality trait of the TT genotype individual is obviously lower than those of the AA and AT genotype individuals. The SNP locus can be used as an auxiliary selection and molecular genetic breeding marker for improving the growth and meat quality traits of poultry.
Example 5 early selection of poultry growth and meat quality traits Using this SNP site
100 full sibling F2 groups of apricot blossom chickens and recessive white rook chickens are purchased, fed to 1 week of age under the same environment, blood samples are extracted to obtain DNA samples, PCR amplification is carried out according to specific primers designed in example 2, and the reaction system is shown in Table 2. The obtained PCR product is sent for testing, and the sequencing result is subjected to genotyping of the mutation site A1514T. The experiments succeeded in typing 100 individuals, as shown in table 5 below.
TABLE 5 genotype frequencies and allele frequencies statistics
In order to verify that the SNP locus can select the growth and the meat quality of the poultry at an early stage, individuals with different types are respectively housed and the feeding conditions are kept consistent. Slaughter measurements were carried out on chickens raised to 6 weeks of age, the initial data being shown in table 6.
TABLE 6A 1514T correlation analysis results with chicken growth, carcass and meat quality traits
Note: the same letter in the same row indicates no significant association and a different letter indicates significant association (P < 0.05).
From the above table 6, it can be seen that the individuals selected and distinguished by the SNP mutation site A1514T have values such as the live weight, breast weight, leg weight, wing weight, carcass weight and the like of AT genotype individuals which are obviously stronger than those of AA and TT genotype individuals, and are also lower in abdominal fat weight and subcutaneous fat thickness, so that the method is suitable for breeding populations with more prominent growth traits; the breast muscle shearing force and leg muscle conductivity rate of the AA genotype individual are obviously superior to those of AT and TT genotypes, and the AA genotype individual is suitable for screening populations for improving meat quality and taste. Experimental data proves that the SNP mutation site A1514T can be applied to early selection of poultry to help select individuals with better growth and carcass traits, so that production cost is saved and genetic progress is accelerated.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> SNP molecular marker related to poultry growth and meat quality traits and application thereof
<130> 2022
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213> Artificial sequence
<400> 1
ccctgacaaa cactgac 17
<210> 2
<211> 17
<212> DNA
<213> Artificial sequence
<400> 2
acacccacaa gaacaag 17
Claims (5)
1. An SNP molecular marker related to poultry growth and meat quality traits, which is characterized in that the SNP molecular marker is positioned in a genomic Z chromosome of galGal6 version, corresponds to a mutation site A1514T in a tenth exon region of a GHR gene, and the genotype of the SNP site is AA, AT and TT.
2. The use of the SNP molecular markers according to claim 1 for the selective breeding of poultry.
3. An early selection method related to poultry growth and meat quality traits, characterized in that poultry growth and meat quality traits are early selected according to the genotype of the SNP molecular marker of claim 1.
4. The early selection method according to claim 3, comprising the steps of:
1) extracting DNA of poultry blood to be detected;
2) carrying out PCR amplification by taking the blood DNA of the poultry to be detected as a template to obtain a PCR product containing a GHR-E10 gene target fragment;
3) detecting the genotype of a mutation site A1514T of the PCR product by adopting a DNA sequencing method;
4) and (3) early selecting the growth and meat quality traits of the poultry based on the genotype of the SNP locus in the step 3), wherein the growth and carcass traits such as the body weight and the like of the AT genotype individual are obviously stronger than those of the AA and TT genotype individuals, and the meat quality traits of the TT genotype individual are obviously lower than those of the AA and AT genotype individuals.
5. The early selection method of claim 4, wherein the nucleotide sequence of the primer used in the PCR amplification of step 3) is:
the upstream primer PCR-F: 5'-CCCTGACAAACACTGAC-3' (SEQ ID NO. 1);
downstream primer PCR-R: 5'-ACACCCACAAGAACAAG-3' (SEQ ID NO. 2).
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