CN114717332B - 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 PDF

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CN114717332B
CN114717332B CN202210434909.2A CN202210434909A CN114717332B CN 114717332 B CN114717332 B CN 114717332B CN 202210434909 A CN202210434909 A CN 202210434909A CN 114717332 B CN114717332 B CN 114717332B
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罗文�
张丹璐
张细权
聂庆华
罗庆斌
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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 a tenth exon sequence of the GHR gene, the mutation site is A1514T, and the genotypes of the SNP site are AA, AT and TT. The SNP molecular marker is obviously related to a plurality of characters such as the growth, carcass, meat quality and the like of poultry, and is a novel molecular marker. Wherein the growth characters and carcass characters of AT genotype individuals such as living weight, chest and leg muscle weight, wing weight, head and foot weight, heart, liver, stomach, adenoma and stomach weight, small intestine length and the like are obviously higher than those of AA and TT genotype individuals, and the subcutaneous fat thickness of AA genotype individuals is obviously higher than those of AT and TT genotype individuals. The meat quality characters such as pectoral muscle shearing force, leg muscle conductivity and the like of the TT genotype individuals are obviously lower than those of the AA and AT genotype individuals. The genotype of the SNP locus is determined to perform early selection on the growth and meat quality traits of the poultry, so that the genetic progress of the weight and meat quality traits of the poultry can be quickened, the production performance of the poultry is improved, the production cost is saved, and the economic benefit of the poultry is improved.

Description

SNP molecular marker related to poultry growth and meat quality traits and application thereof
Technical Field
The invention belongs to the technical field of molecular biotechnology and molecular marking, 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 acts, the first step is to bind to GH receptors on the surface of target cells and then signal into the cells via GHR, promoting the activity of the associated pathways of intracellular growth. Growth Hormone Receptor (GHR) is a key gene for GH to regulate cell growth. The Growth Hormone Receptor (GHR) gene is also reported to be involved in fat deposition in chickens. During the growth and development of animals, growth hormone must bind to growth hormone receptors in order to function. For cattle, when the base sequence of the GHR gene is mutated, the normal function of GH is influenced, and thus, various properties such as milk production, meat production and the like are influenced. Research shows that when the secretion of growth hormone is increased, the growth performance of the broiler chickens can be improved, the metabolism of animal substances and energy is promoted, and the growth and development process is promoted. Meanwhile, GH gene is an important functional gene for poultry and plays a key role in the growth and development of poultry.
GHR is the basis for GH to function normally and has close relationship with growth and development of animals. GHR is an important transmembrane glycoprotein encoded by a single gene and contains 620 amino acids, and when the organs in the animal body contain enough GHR, the GHR can normally function as prolactin, cytokines, growth hormone, erythropoietin and the like. GHR exists in most cells of the organism, plays a role in binding with GH to play a physiological role, is particularly prominent in liver, and is found to be the organ with the greatest expression of chicken GHR in the research of chicken GHR. Besides the liver, the GHR gene is expressed in skin, heart, muscle, lung, kidney, testis, ovary, adrenal gland, brain, lymphoid tissue, etc. However, different positions of the GHR gene can also play different functions in different organs, and different regulation methods are available. Studies have shown that GH gene expression can have an effect on birth weight, weaning weight and 1 year weight in cattle. The transmembrane structure of the chicken GHR receptor is located at 24 amino acid residues 238-261, the GHR gene contains 592 amino acid residues, and a 16 amino acid signal peptide is found at the N terminal end of the GHR gene. The amino acid sequence of GHR in chickens has a relatively low homology to mammals, for example 53% homology to chickens and rabbits, and 58% homology to mice. In contrast, the homology of GHR is relatively high between mammals, for example, the homology of human, mouse and rabbit reaches 70% and 84%, respectively, and the three are relatively similar in structure, but all are composed of three parts of extracellular structure, intracellular structure and transmembrane structure. Wherein the extracellular structure is responsible for binding to the extra-membranous ligand, and the disulfide bond formed by the 7 cysteines present in the extracellular structure is capable of allowing the GHR to retain a specific spatial structure outside the cell without deformation.
With the continuous development of molecular marking technology and molecular number genetics, the application of molecular genetic markers for marking auxiliary selection can greatly shorten the generation interval of breeding and accelerate the genetic progress of breeding. The single nucleotide polymorphism (Single Nucleotide Polymorphisms, SNPs) mainly refers to DNA sequence polymorphism caused by single nucleotide variation at genome level, has the characteristics of easy statistics, wide distribution, good genetic stability and the like, is the most widely applied genetic marking technology at present, and plays an important role in the research field of animal genetic breeding. The local varieties in China are rich in resources, and most local varieties of poultry have excellent characters of strong stress resistance, weak nest-forming 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 larger market demand, but the weight and growth speed of poultry are excessively selected by the breeding industry in recent decades, so that fat deposition in the poultry is excessive, and the meat quality and the character of the poultry are affected. The poultry GHR gene is positioned on chromosome 5, has a gene length of 87kb and comprises 10 exons, finds SNPs related to the growth and meat quality traits of the poultry, and can be applied to molecular breeding, so that the genetic progress can be quickened, the production cost can be saved, and valuable information can be provided for the breeding of the poultry.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an SNP molecular marker related to the growth and meat quality traits of poultry, and establish a marker genotype detection method, which is applied to the breeding of poultry to improve the carcass traits of the poultry.
In a first aspect, the invention provides an SNP molecular marker related to the growth and meat quality traits of poultry, wherein the SNP molecular marker is positioned in a Z chromosome of a galGal th edition genome, a mutation site in a tenth exon sequence corresponding to a GHR gene is A1514T, and genotypes of the SNP site are AA, AT and TT.
A second aspect of the invention is to provide an early selection method associated 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 poultry GHR gene to be detected, and individuals with expected carcass traits conforming to expected targets are obtained for feeding, so that the overall feeding cost is reduced, and the output value is improved.
Further, the early selection method includes the steps of:
1) Extracting DNA of poultry blood to be detected;
2) Taking blood DNA of poultry to be detected as a template, and carrying out PCR amplification to obtain a PCR product containing a target fragment of the GHR gene;
3) Detecting the genotype of a mutation site A1514T of the PCR product by adopting a DNA sequencing method;
4) Early selecting the growth and meat quality traits of the poultry based on the genotypes of the SNP loci in the step 3), wherein the growth and carcass traits such as the weight of AT genotype individuals are obviously stronger than those of AA and TT genotype individuals, and the meat quality traits of TT genotype individuals are obviously lower than those of AA and AT genotype individuals. During breeding, individuals retaining AT genotype 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:
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, a mutation site A1514T in a poultry GHR gene exon 10 sequence is related to growth and meat quality traits of poultry, is a novel molecular marker. The genotype of the SNP locus is determined to perform early selection on the growth and meat quality traits of poultry, so that the growth and meat quality traits of meat poultry can be improved, the production cost is saved, the genetic progress is quickened, and the SNP locus is better applied to poultry breeding and has great economic application value and scientific research value.
Drawings
FIG. 1 is a graph showing the genotyping result of the mutation site A1514T in the GHR gene.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with specific embodiments, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 extraction of blood DNA from Emblica officinalis chicken X recessive white Rockwell chicken F2 Generation resource group isotactic cell line
The DNA sample is a DNA sample of a isotactic F2 resource population of the apricot flower chickens and the recessive white roc chickens stored in the laboratory. The recessive white rock chicken is a fast-growing broiler chicken, and the apricot-colored chicken is a local variety of broiler chicken in China. The second-generation broiler bred from apricot-flower chickens and recessive white rocky chickens adopts a raising mode of raising in a flat mode, and is fed with corn soybean feed meeting international formula standards. Samples were slaughtered at 90 days of age and their shank length, head width, chest depth, body length, chest angle width, slaughter weight, cortex thickness, evisceration weight, bore weight, pectoral muscle weight, leg muscle weight, feather weight, abdominal fat weight, head and neck weight, heart weight, liver weight, stomach weight and small intestine length were recorded. Wherein the length of the shin, the width of the head, the width of the chest and the depth of the chest and the length of the body are measured by a vernier caliper; chest angle is measured by a chest angle device; subcutaneous fat thickness was measured on the back near the tail, fat width was measured between the leg and pectoral muscle, and both properties were measured with vernier calipers.
Genomic DNA from all individualsPLANT MINI KIT (Qiagen, hilden, calif.; cat # 69104) kit protocol blood DNA was extracted, assayed for quality and concentration, diluted to 50 ng/. Mu.L, and stored at 4℃for further use.
Example 2 SNP detection and DNA pool sequencing
1. Primer design and specificity detection:
Primer sequences for amplifying the GHR gene were designed based on the GHR gene sequences published on GeneBank as shown in Table 1. The designed primer pair sequence is sent to a biological company (Shanghai) for synthesis, and the primer pair is used for carrying out PCR amplification on GHR gene standard substances, and the specificity of the primers is tested. The SNP locus is positioned at the 116 th base of the amplified 689bp fragment sequence.
Primer information for SNP screening of Table 1 GHR
2. Sequencing by a DNA mixing pool:
10 DNA samples are randomly selected from the total extracted DNA samples to construct a mixed pool, the mixed pool samples are subjected to PCR amplification, the used primers are the same as the primer pairs used in SNP detection, and the reaction system is shown in Table 2.
TABLE 2 PCR reaction system
PCR reaction procedure: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, annealing at 52℃for 30s, elongation at 72℃for 1min,37 cycles; extending for 10min at 72 ℃; preserving at 4 ℃.
And (3) sequencing the obtained PCR product to a biological company, and detecting the sequencing result to obtain the mutation site A1514T in the tenth exon sequence of the poultry GHR gene, wherein the sequencing result analysis is shown in the attached figure 1.
Example 3 determination of genotype of GHR mutation site A1514T
The whole 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 the target fragment of the GHR gene. The PCR products were sequenced separately, and the genotype of each sequencing result was analyzed, and the typing results are shown in FIG. 1. 199 individuals were successfully typed in this experimental example, and then each genotype was statistically analyzed to obtain the genotype frequency and allele frequency for that locus. The statistical structure of the gene and genotype frequency of the SNP mutation site A1514T is shown in the following table 2.
TABLE 3 genotype frequency allele frequencies for GHR Gene mutation site A1514T
Wherein:
(1) Genotype frequency refers to the ratio of the number of individuals of a certain genotype to the total number of genotypes in a population:
genotype frequency = total number of genotypes/total number of populations x 100%;
(2) Gene frequency refers to the ratio of a gene to all genes at the same site in a population:
Gene frequency = number of genes per total number of genes at the same locus in a population x 100%.
As can be seen from the structure of Table 3, the AA genotype is the dominant genotype of the chicken population to be tested.
Example 4 correlation analysis of SNP mutation site A1514T with poultry growth and meat quality traits
The correlation analysis of different genotypes of the GHR gene with the F2 population growth and carcass traits was performed using SPSS software, and the results are expressed as (mean ± standard error). Significance analysis was performed with t-test, P <0.05 indicated that the difference was significant, and P <0.01 indicated that the difference was extremely significant. The correlation analysis results are shown in table 4 below.
TABLE 4 analysis of the relationship between GHR Gene mutation site A1514T and poultry growth, carcass and meat quality traits
Note that: the same letter in the same row indicates that the association is not significant and the different letters indicate that the association is significant (P < 0.05).
From the table it can be seen that the mutation site is significantly associated with several traits like fowl liveweight, chest meat weight, leg meat weight, wing weight, head and neck weight, foot weight, heart liver muscle stomach adenoma, small intestine length, carcass weight, chest angle, total bore-free weight, semi bore-free weight, pectoral muscle shear force, leg muscle conductivity, body weight 28, body weight 35, body weight 42, shank length 42, shank diameter 42, body weight 49, shank length 49, shank diameter 49 etc. (P < 0.05). Wherein the weight and other growth and carcass traits of AT genotype individuals are obviously stronger than those of AA and TT genotype individuals, and the meat quality traits of TT genotype individuals are obviously lower than those of 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 the SNP locus
100 Groups of apricot-flowered chickens and recessive white-rocky chickens of the whole sibling F2 are purchased, the apricot-flowered chickens and the recessive white-rocky chickens are fed to the age of 1 week under the same environment, blood samples are extracted, DNA samples are obtained, PCR amplification is carried out according to specific primers designed in example 2, and a reaction system is shown in Table 2. And (3) sending and measuring the obtained PCR product, and carrying out genotyping of the mutation site A1514T on the sequencing result. The experiment was successful 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 meat quality traits of poultry in early stage, individuals with different types are respectively bred in a captive mode, and the breeding conditions are kept consistent. Chickens raised to 6 weeks of age were slaughtered and assayed, and initial data are shown in Table 6.
TABLE 6 correlation analysis of A1514T with chicken growth, carcass and meat quality traits
Note that: the same letter in the same row indicates that the association is not significant and the different letters indicate that the association is significant (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 significantly stronger values of the AT genotype individuals such as the live weight, chest weight, leg weight, wing weight, carcass weight and the like than those of the AA and TT genotype individuals, and lower abdominal fat weight and subcutaneous fat thickness, and are suitable for cultivating populations with more prominent growth traits; the pectoral muscle shearing force and the leg muscle conductivity rate of the AA genotype individual are obviously superior to those of the AT and TT genotypes, and the AA genotype individual is suitable for screening the population for improving the meat quality and taste. Experimental data prove that SNP mutation site A1514T can be applied to early selection of poultry to help select individuals with better growth and carcass traits, so as to save production cost and accelerate genetic progress.
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 characteristics 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> Agricultural university of south China
<120> SNP molecular markers related to poultry growth and meat quality traits and uses 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 (2)

1. The application of the primer pair for specifically amplifying SNP molecular markers in breeding related to growth and meat quality traits of poultry is characterized in that the poultry is a filial generation of apricot-pattern chickens and recessive white-roc chickens, the nucleotide sequences of the primer pair are respectively shown as SEQ ID No.1 and SEQ ID No.2, the SNP molecular markers are positioned AT the 116 th base of the nucleotide sequence of an amplified product, the mutation type is A & gtT, the genotype of the SNP molecular markers is AA, AT or TT, the weight growth and carcass traits of AT genotype individuals are obviously stronger than those of AA and TT genotype individuals, and the meat quality traits of TT genotype individuals are obviously lower than those of AA and AT genotype individuals.
2. An early selection method related to the growth and meat quality traits of poultry, which is characterized in that the poultry is a filial generation of apricot-flowered chicken and recessive white rock chicken, the early selection is carried out on the growth and meat quality traits of the poultry according to the genotype of SNP molecular markers, and the method comprises the following steps:
1) Extracting DNA of poultry blood to be detected;
2) Taking blood DNA of poultry to be detected as a template, and carrying out PCR amplification to obtain a product, wherein the nucleotide sequences of primer pairs for PCR amplification are respectively shown as SEQ ID No.1 and SEQ ID No. 2;
3) Detecting PCR amplification products by adopting a DNA sequencing method, wherein the SNP molecular marker is positioned AT the 116 th base of the nucleotide sequence of the amplification products, the mutant type is A & gtT, and the genotype of the SNP molecular marker is AA, AT or TT;
4) Early selection of the growth and meat quality traits of the poultry based on the genotypes of the SNP molecular markers in the step 3), wherein the growth and carcass traits such as the weight of AT genotype individuals are obviously stronger than those of AA and TT genotype individuals, and the meat quality traits of TT genotype individuals are obviously lower than those of AA and AT genotype individuals.
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CN104593363A (en) * 2015-01-13 2015-05-06 山东大学 Excellent slaughter trait molecular genetic marker of broiler chicken and application of excellent slaughter trait molecular genetic marker
CN106011273A (en) * 2016-07-13 2016-10-12 甘肃省畜牧兽医研究所 Method for detecting Zaosheng cattle GH gene mononucleotide polymorphism

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WO2007129219A2 (en) * 2006-01-13 2007-11-15 The Governors Of The University Of Alberta Polymorphisms in growth hormone receptor, ghrelin, leptin, neuropeptide y, and uncoupling protein 2 genes and their associations with measures of performance and carcass merit in beef cattle
WO2010077832A1 (en) * 2008-12-15 2010-07-08 Wisconsin Alumni Research Foundation Methods and compositions for testing and breeding cattle for improved fertility and embryonic survival
CN109207609B (en) * 2018-10-17 2022-03-22 佛山科学技术学院 SNP (Single nucleotide polymorphism) related to chicken growth traits and application thereof
CN109234405B (en) * 2018-10-17 2021-04-20 华南农业大学 SNP molecular marker related to chicken growth traits and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104593363A (en) * 2015-01-13 2015-05-06 山东大学 Excellent slaughter trait molecular genetic marker of broiler chicken and application of excellent slaughter trait molecular genetic marker
CN106011273A (en) * 2016-07-13 2016-10-12 甘肃省畜牧兽医研究所 Method for detecting Zaosheng cattle GH gene mononucleotide polymorphism

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