CN114438231A - CRELD1 gene molecular marker related to chicken carcass traits and application - Google Patents
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Abstract
The invention discloses a CRELD1 gene molecular marker related to chicken carcass traits and application thereof, and relates to the technical field of biology. The molecular marker is represented by a sequence shown in SEQ ID NO. 1, wherein the sequence exists NC-052543.1: g.11683326G > A and a sequence shown in SEQ ID NO. 1, wherein the sequence exists NC-052543.1: g.11683378G > A, the sequence shown in SEQ ID NO. 1, wherein the sequence exists NC-052543.1: g.11683497T > C, and the sequence shown in SEQ ID NO. 1, wherein the sequence exists NC-052543.1: g.11683588A > G. The 4 molecular markers are all obviously related to the carcass traits of chickens. Therefore, the method can accurately identify the chicken carcass traits and provide a new molecular marker for molecular marker-assisted selective breeding.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a CRELD1 gene molecular marker related to chicken carcass traits and application thereof.
Background
Single Nucleotide Polymorphism (SNP) refers to a Polymorphism of a genomic DNA sequence caused by insertion, deletion, transversion, conversion, etc. of a Single Nucleotide at the genomic level. The SNP is the most common animal genetic variation, widely exists in animal genomes, and has the characteristics of stable inheritance, easy detection and the like. In animal production practice, SNP can be used for Molecular marker-assisted Selection (MAS) so as to break through the bottleneck of traditional breeding and improve the accuracy of seed Selection and the breeding effect of target traits.
The Cysteine-rich protein domain 1 gene (Cysteine rich with EGF like domains 1, CRELD1) of the epidermal growth factor family is the first gene found to be related to the onset of congenital heart atrial-ventricular septal defect, and the encoded CRELD1 protein is an important regulator of the embryonic development process. The important role of CRELD1 in the process of embryonic development also has certain influence on the phenotype of adult livestock and poultry carcass traits. However, no report about the relevance of the CRELD1 gene to SNP molecular markers related to poultry carcass traits exists at present.
Disclosure of Invention
The invention aims to provide a CRELD1 gene molecular marker related to chicken carcass traits and application thereof, which are used for solving the problems in the prior art, discovering the SNP site of a CRELD1 gene sequence, analyzing the SNP site in association with the chicken carcass traits and providing a new SNP molecular marker for MAS
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a molecular marker related to chicken carcass traits, which is SNP1, SNP2, SNP3 and SNP4, wherein the SNP1 is that a sequence shown as SEQ ID NO. 1 has NC-052543.1: g.11683326G > A; the SNP2 is a sequence shown by SEQ ID NO. 1 and exists in NC-052543.1: g.11683378G > A; the SNP3 is the sequence shown in SEQ ID NO. 1 and exists in NC-052543.1: g.11683497T > C; the SNP4 is a sequence shown in SEQ ID NO. 1 and exists in NC-052543.1: g.11683588A > G.
Preferably, the SNPs 1 are classified into GG, GA and AA genotypes; the SNP2 is divided into GG, GA and AA genotypes; the SNP3 is divided into TT, TC and CC genotypes; the SNP4 is classified into AA, AG and GG genotypes.
The invention also provides a primer for amplifying the molecular marker, which is an upstream primer shown as SEQ ID NO. 2 and a downstream primer shown as SEQ ID NO. 3.
The invention also provides a kit for identifying the carcass traits of chickens, which comprises the primer.
The invention also provides a method for identifying the carcass traits of chickens, which comprises the following steps:
(1) taking the genome DNA of the chicken to be detected as a template, and amplifying a target fragment by using the primer to obtain an amplification product;
(2) sequencing the obtained amplification product, detecting the genotype of the corresponding single nucleotide polymorphism site, and judging the chicken carcass traits according to the genotype.
Preferably, the amplification system is: template DNA 2. mu.L, 2 XEs Taq MasterMix (Dye) 25. mu.L, upstream primer 2. mu.L, downstream primer 2. mu.L and ddH2O 19μL。
Preferably, the reaction procedure is: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; final extension at 72 ℃ for 3 min; storing at 4 deg.C.
Preferably, the carcass traits include live body weight, shin length, cloaca skin yellowness value, breast muscle weight, carcass weight, shin circumference and half-bore ratio.
The invention also provides application of the molecular marker or the primer pair or the kit or the method in chicken breeding.
Preferably, the method is applied to breeding of chicken carcass traits.
The invention discloses the following technical effects:
according to the invention, through analyzing the CRELD1 gene, the gene is found to have a plurality of SNP sites which are obviously related to the chicken carcass traits, a new SNP molecular marker is provided for MAS, and the following steps are verified through experiments: the site of the molecular marker NC-052543.1: g.11683326G > A is obviously related to the values of chest muscle weight, live body weight, carcass weight, shin length, shin circumference and cloaca skin yellowness (p is less than 0.05); the molecular marker NC-052543.1: g.11683378G > A site has obvious correlation with the half-bore rate (p < 0.05); the molecular marker NC-052543.1: g.11683497T > C site has a significant correlation with the half-bore rate (p < 0.05); the molecular marker NC-052543.1: g.11683588A > G locus has significant correlation with the half-bore ratio and the tibioperineum (p < 0.05). Therefore, the invention discovers that the 4 molecular markers are all related to the chicken carcass traits, and can accurately identify the chicken carcass traits, thereby providing scientific basis for chicken breeding.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of the CRELD1 primer pairing position and product length;
FIG. 2 is a diagram of the genotyping of SNP sites of the CRELD1 gene sequence.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
1. Materials and methods
1.1 animal samples
The test animals were 450 spotted-brown chickens of 80 days old, and 409 spotted-brown chickens with phenotype records. By collecting 2mL of subcutaneous venous blood, the sample was stored at-80 ℃ and used as a DNA extraction sample. Meanwhile, carcass traits such as subcutaneous fat thickness, intramuscular fat width, full bore weight, half bore weight, abdominal fat weight, leg weight, pectoral muscle rate, leg ratio, abdominal fat rate, half bore rate, full bore rate, lean meat rate, slaughter rate and the like of a selected colony are recorded.
1.2 Primary reagents
Blood sample DNA extraction kit (brand: OMEGA; cat # D3392; Feiyang bioengineering, Guangzhou), 2 XEs Taq MasterMix (Dye) (brand: Kangshi; cat # CW 0690M; Kangshi Biotechnology, Inc.), DNA marker (brand: Novozan; cat # MD 101; Jiangsu Novozan Biotechnology, Inc.), high purity low electroosmosis agarose (brand: Tokyo; cat # TSJ 001; Beijing Tokyo Biotechnology, Inc.).
1.3 Experimental methods
1.3.1 primer design
According to the sequence of Red Chicken (Gallus) CRELD1 Gene (Gene ID:100858613) published by NCBI (national Center for Biotechnology Information Search database), primers were designed using NCBI's Primer-BLAST tool, and Primer synthesis services were provided by Ongchow Biotechnology, Inc. The information about the primer sequences is shown in Table 1, and the pairing positions of the primers on the CRELD1 gene are shown in FIG. 1.
TABLE 1 PCR amplification primer sequences
1.3.2 blood sample DNA extraction
And (4) extracting the DNA of the blood sample by referring to an operation manual of the blood sample DNA extraction kit.
1.3.3 PCR amplification of partial sequence of CRELD1 Gene
The genomic DNA of the 409 individual blood samples was used as a template, and the following reaction system was followed: template DNA 2. mu.L, 2 XEs Taq MasterMix (Dye) 25. mu.L, upstream primer 2. mu.L, downstream primer 2. mu. L, ddH2O 19μL。
Reaction procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; final extension at 72 ℃ for 3 min; storing at 4 ℃. PCR products were subjected to Sanger sequencing by Gene technology, Inc., Tianyihui, Guangzhou.
1.3.4SNP determination and genotyping
Analysis of the sequence peak patterns was performed on the Sanger sequencing results of the PCR products using the SeqMan tool of DNAstar software to determine potential SNP sites, and sequencing data for each sample was aligned by this tool for genotyping.
1.3.5 genotype correlation with carcass traits
The data of individual carcass traits corresponding to SNP sites and genotypes are subjected to correlation analysis by adopting a SAS 9.4GLM program package.
2 results
2.1 PCR amplification and SNP screening of CRELD1 Gene sequences
Selecting blood sample DNA of 409 individuals of the domestic chicken as a template to carry out PCR amplification, carrying out Sanger sequencing on an obtained PCR product (the nucleotide sequence is shown as SEQ ID NO: 1), carrying out comparison analysis on a peak diagram after sequencing, and detecting 5 SNP sites in total, wherein 4 sites are associated with the chicken carcass traits and respectively comprise: NC-052543.1 g.11683326G > A, NC-052543.1 g.11683378G > A, NC-052543.1 g.11683497T > C and NC-052543.1 g.11683588A > G, as shown in FIG. 2.
SEQ ID NO:1
ACTATGAGGCATCACGCAACCAAAGCCAGCTGCTGTGTGCCGGTAAGCACCGTGCCGTGCTGCGCTGAGGGCTCTGTGCCCACCCCCTTGTGCCCACCACCCTGTGCTCACCGCCCTGTGTCCACCCCTGCAGAGTGTTACCCTGCCTGTGGGCGCTGCACGGGGCCGGAGGACTCCAGCTGCCTGCGCTGCAAGAGGGGCTGGGTGCTGCACGAGCACCGCTGCATCGGTCGGTGCACGGGGACGTGGGGCTGGATTTTGGGGTCGGGGTCCCGGGGAGCGCTGTGGAATCGTTCCCTCTGATCCACAGATATTGATGAGTGCGGCACGGAGATGGCACACTGCAGGGCCAACCAGTTTTGCGTCAACACTGAAGGCTCCTACGAGTGCCGAGGTGCGAGGGCAGCGGGGTAGCGATGGGATGGCAGTGTTGGGTGGCCCCCTTCAGCTGTGGCAGCCCCCCATCTTCCTTTCTGTCCCCTCCTCAATACGCCGCAGACTGCTCCACCGCCTGCATCGGCTGCATGGGCGCAGGGCCGGCACGTTGCAAGAAGTGCAACAAGGGCTACCGGCGGGACGGGGCCAAGTGCCTGGGTGAGTGCTCTTGGCACGCCCGTAGGGGGGACGCGGTGCCCGGTGCCCCCGCGCCGCTACCTGGGTGCTCCCTGCAGACGTGGATGAGTGCACCAGCAGCGAGGAGCCCATCTGCACGGGGCTGCAGGAGGTGTGCGAGAACACGGAGGGCGGCTATCGCTGCGTCTGCGCCCCGGGCCACGTGCGCAGGGATGGGCAGTGCGTGGAGGACAAACCCCCCGGTATGGCACGGGATTGGGGAGGGGGGCGCGGAGGGATGCCCTCACCCACCATTGTCACCCTTGCAGACGCCCCGGAGAAGGGTTTCTTTGACGACGTGACG
2.2 analysis of association of SNP site of CRELD1 Gene sequence with carcass traits
The above 5 SNP sites were correlated with carcass traits (thick subcutaneous fat, wide intramuscular fat, full bore weight, half bore weight, abdominal fat weight, leg weight, breast muscle weight, pectoral muscle rate, leg ratio, abdominal fat rate, half bore rate, full bore rate, lean meat rate, slaughter rate, live body weight, shin length, shin girth and carcass cloaca skin color, shoulder skin color, hip skin color, abdominal skin color, chest leg skin color, shin skin color and abdominal fat color yellowness value).
As shown in Table 2, the results show that the NC-052543.1: g.11683326G > A site has significant correlation (p <0.05) with values of breast muscle weight, live body weight, carcass weight, shin length, shin girth and cloaca skin yellowness, wherein the live body weight of AA genotype individuals and GA genotype individuals is significantly higher than that of GG genotype individuals (p <0.05), but there is no significant difference between AA and GA (p > 0.05); the weight of pectoral muscle, the weight of living body and the weight of carcass of the GA genotype individual are obviously higher than those of the GG genotype individual (p <0.05), and the length and circumference of shin are obviously higher than those of the AA genotype individual (p < 0.05); cloacal skin yellowness values were significantly higher for AA genotype individuals than for GG genotype individuals (p < 0.05).
As shown in Table 3, the results show that NC _052543.1: g.11683378G > A site has a significant correlation with the half-bore rate (p <0.05), wherein the half-bore rate of the GG genotype individuals and the GA genotype individuals is significantly higher than that of the AA genotype individuals (p <0.05), but there is no significant difference between the GG and the GA (p > 0.05).
As shown in Table 4, the results showed that there was a significant correlation (p <0.05) between the NC-052543.1: g.11683497T > C locus and the half-bore rate. Wherein the half-bore rate of individuals with CC and TC genotypes is significantly higher than that of individuals with TT genotypes (p <0.05), but there is no significant difference between CC and TC (p > 0.05).
As shown in Table 5, the results show that the NC-052543.1: g.11683588A > G locus has a significant correlation with the half-bore ratio and the tibial circumference (p < 0.05). Wherein the half-bore rate and the tibiod circumference of the GG genotype individuals and the AG genotype individuals are both significantly higher than those of the AA genotype individuals, and there is no significant difference between GG and AG (p > 0.05).
In addition to the 4SNP sites mentioned above, the association of other sites within the amplified fragment with the carcass trait did not reach a significant level (p > 0.05).
TABLE 2 NC-052543.1 g.11683326G > A sites associated with carcass traits
TABLE 3 NC-052543.1 g.11683378G > A site associated with carcass traits
TABLE 4 NC-052543.1 g.11683497T > C site associated with carcass traits
TABLE 5 NC-052543.1 g.11683588A > G site associated with carcass traits
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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tccacccctg cagagtgtta ccctgcctgt gggcgctgca cggggccgga ggactccagc 180
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Claims (10)
1. The molecular marker related to the chicken carcass trait is characterized in that the molecular marker is SNP1, SNP2, SNP3 and SNP4, wherein the SNP1 is NC-052543.1: g.11683326G > A in a sequence shown as SEQ ID NO. 1; the SNP2 is a sequence shown by SEQ ID NO. 1 and exists in NC-052543.1: g.11683378G > A; the SNP3 is the sequence shown in SEQ ID NO. 1 and exists in NC-052543.1: g.11683497T > C; the SNP4 is a sequence shown in SEQ ID NO. 1 and exists in NC-052543.1: g.11683588A > G.
2. The molecular marker of claim 1, wherein the SNP1 is classified into a GG, GA, and AA genotype; the SNP2 is divided into GG, GA and AA genotypes; the SNP3 is divided into TT, TC and CC genotypes; the SNP4 is classified into AA, AG and GG genotypes.
3. A primer for amplifying the molecular marker of claim 1, which is an upstream primer shown as SEQ ID NO. 2 and a downstream primer shown as SEQ ID NO. 3.
4. A kit for identifying a carcass trait in a chicken comprising the primer of claim 3.
5. A method of identifying a carcass trait in a chicken comprising the steps of:
(1) taking the genome DNA of a chicken to be detected as a template, and amplifying a target fragment by using the primer of claim 3 to obtain an amplification product;
(2) sequencing the obtained amplification product, detecting the genotype of the corresponding single nucleotide polymorphism site, and judging the chicken carcass traits according to the genotype.
6. The method of identifying a chicken carcass trait of claim 5 wherein the amplification system is: template DNA 2. mu.L, 2 XEs Taq MasterMix (Dye) 25. mu.L, upstream primer 2. mu.L, downstream primer 2. mu.L and ddH2O 19μL。
7. The method of identifying a chicken carcass trait of claim 5 wherein the reaction program is: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, and 32 cycles; final extension at 72 ℃ for 3 min; storing at 4 ℃.
8. The method of identifying chicken carcass traits as claimed in claim 5, characterized in that the carcass traits comprise live body weight, shin length, cloaca skin yellowness value, breast muscle weight, carcass weight, shin circumference and half-bore ratio.
9. Use of the molecular marker of claim 1 or the primer pair of claim 3 or the kit of claim 4 or the method of claim 5 in chicken breeding.
10. The use of claim 9 in the breeding of chicken carcass traits.
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