CN114622020B - KLHL31 gene molecular marker related to chicken growth traits and application thereof - Google Patents
KLHL31 gene molecular marker related to chicken growth traits and application thereof Download PDFInfo
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Abstract
The invention discloses a KLHL31 gene molecular marker related to chicken growth traits and application thereof, relating to the technical field of biology. The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, the molecular marker has two SNP sites, SNP1 is NC-052534.1: g.87828628G > C, and SNP2 is NC-052534.1: g.87828969C > T. According to the invention, through analyzing the KLHL31 gene, a plurality of SNP sites which are obviously related to the growth traits of chickens are found in an amplified fragment region of the gene, and through experimental verification, the SNP1 site is obviously related to the weight of 91-day-old chickens (p is less than 0.05); the SNP2 site was significantly correlated with 77-day-old body weight and 84-day-old body weight average (p < 0.05).
Description
Technical Field
The invention relates to the technical field of biology, in particular to a KLHL31 gene molecular marker related to chicken growth traits and application thereof.
Background
Single Nucleotide Polymorphism (SNP) refers to a Polymorphism of a DNA sequence caused by variation of Single Nucleotide insertion, deletion, transversion, and transition at the genome 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), and the seed Selection accuracy and the breeding effect are improved. Finding out effective molecular markers related to sexual maturity can provide favorable technical support for molecular marker-assisted selective breeding of chicken sexual growth traits.
KLHL31(Kelch like family member 31) is a member of the Kelch-like family of vertebrates and encodes a protein containing a conserved BTB domain and a tandem repeat of the Kelch domain.
Disclosure of Invention
The invention aims to provide a molecular marker of a KLHL31 gene related to chicken growth traits and application thereof, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a KLHL31 gene molecular marker related to chicken growth traits, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, the molecular marker has two SNP sites, the SNP1 is NC-052534.1: g.87828628G > C, and the SNP2 is NC-052534.1: g.87828969C > T.
Further, the genotype of the SNP1 includes GG, GC, and CC; the genotype of the SNP2 includes CC, CT, and TT.
The invention also provides a primer pair for identifying the growth traits of the chicken, and the sequence of the primer pair is shown as SEQ ID NO. 2-3.
The invention also provides a method for identifying the growth traits of chickens by using the molecular marker, which comprises the following steps:
(1) extracting the genome DNA of the chicken to be detected, and carrying out PCR amplification by using the primer pair to obtain an amplification product;
(2) sequencing the amplification product, detecting the genotype of SNP1 site, and when the genotype of SNP1 site is GG, the size of the body is 91 days old; the genotype of SNP2 site was examined, and when the genotype of SNP2 site was CC, the body weight at 77 days old and the body weight at 84 days old were significant.
Further, in the step (1), the sequence of the primer pair amplified by the PCR is the sequence shown in SEQ ID NO. 2-3.
Further, in step (1), the amplification system is: 2 mu L of template DNA, 25 mu L of 2 xRapid Taq Master Mix, 2 mu L of upstream primer and 2 mu L, ddH of downstream primer 2 O 19μL。
Further, in step (1), the amplification reaction procedure is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 15s, and 35 cycles; completely extending for 3min at 72 ℃; storing at 4 ℃.
The invention also provides a kit for identifying the growth traits of the chickens, which comprises the primer pair.
The invention also provides application of the KLHL31 gene molecular marker, the primer pair or the kit related to the chicken growth traits in chicken genetic breeding.
Further, the chicken genetic breeding is the breeding of chicken varieties with high growth speed.
The invention discloses the following technical effects:
according to the invention, through analyzing the KLHL31 gene, a plurality of SNP loci which are obviously related to chicken growth traits are found in an amplified fragment region of the gene, a new SNP molecular marker is provided for molecular marker assisted selection, and through experimental verification, the molecular marker NC-052534.1: g.87828628G > C locus has obvious correlation with 91-day-old body weight (p <0.05), wherein the 91-day-old body weight of a GG homozygous genotype individual is obviously larger than that of a GC heterozygous genotype individual (p <0.05), and the CC homozygous genotype individual has no obvious difference with the GG homozygous genotype individual and the GC heterozygous genotype individual (p > 0.05); NC-052534.1: g.87828969C > T locus has obvious correlation (p <0.05) with weight of 77-day-old and 84-day-old individuals, wherein the weight of 77-day-old and 84-day-old individuals of CC homozygous genotype is obviously larger than that of TT homozygous genotype (p <0.05), and the weight of CT heterozygous genotype has no obvious difference (p >0.05) with the CC homozygous genotype and the TT homozygous genotype. The association of other SNP loci with chicken growth traits does not reach a significant level (p > 0.05). Therefore, the molecular marker provided by the application can accurately identify the growth traits of the chickens, so as to provide scientific data for breeding the chickens.
Drawings
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 showing the pairing position and product length of KLHL31 primer;
FIG. 2 is a genotype chart of SNP site of KLHL31 gene amplification fragment region.
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 intervening value, to the extent 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 herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. 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 description and examples are intended to be illustrative 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
328 female Ningdu Sanhuang chickens were selected, which were 300 days old. 2mL of subcutaneous venous blood was collected and stored at-80 ℃ and used as a DNA extraction sample. Meanwhile, family information and growth traits of the selected population are recorded.
1.2 Primary reagents
Blood sample DNA extraction kit (brand: OMEGA; cat # D3392; Feiyang bioengineering, Guangzhou), 2 × Rapid Taq Master Mix (brand: Novozan; cat # P222-01; Nevozan Biotech, Inc., Jiangsu), DNA marker (brand: Novozan; cat # MD 101; Nevozan Biotech, Inc., Jiangsu), high purity low electroosmosis agarose (brand: cat # TSJ 001; Beijing Stratake Biotechnology, Inc.).
1.3 Experimental methods
1.3.1 primer design
According to the sequence of the Red Chicken (Gallus) KLHL31 gene (GeneID:421893) 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 Guangzhou Tianyihui Genbank technology, Inc. The information on the primer sequences is shown in Table 1, and the positions of the primers paired with the KLHL31 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 the exon sequence of the KLHL31 Gene
The genomic DNA of the blood samples of the 328 individuals was used as a template, and PCR amplification was carried out by following the following reaction system: template DNA 2. mu.L, 2 × Rapid Taq Master Mix 25. mu.L, upstream primer 2. mu.L, downstream primer 2. mu. L, ddH 2 O 19μL。
Reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 15s, and 35 cycles; completely extending for 3min at 72 ℃; storing at 4 ℃. PCR products were subjected to Sanger sequencing by Gene technology, Inc., Tianyihui, Guangzhou.
1.3.4 SNPs determination and genotyping
Analysis of the sequence peak map 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 and growth trait Association analysis
And carrying out correlation analysis on the SNPs locus and the growth character data of the individual corresponding to the genotype by adopting an SAS 9.4 GLM program package.
2. Results
2.1 PCR amplification and SNP screening of the amplified fragment region sequence of the KLHL31 Gene
328 individuals are selected, PCR amplification is carried out by taking blood sample DNA of each individual as a template, the obtained PCR product (the nucleotide sequence is shown as SEQ ID NO: 1) is subjected to Sanger sequencing, the peak images after sequencing are compared and analyzed, 3 SNP sites are detected in total, wherein 2 sites are obviously related to growth traits and are NC-052534.1: g.87828628G > C and NC-052534.1: g.87828969C > T. As shown in fig. 2.
SEQ ID NO:1：
GTCGTCACCCAGCAGAAGAAGAAAAAAATTCCTGGTACTCACAGGCAGCCACCATTCCTTTCTATAATTAAATTACCATTTCTTTATGCGCCGTGCACTTAAGGGAAAATCAATGTGCTGAGAGTTCAGATTGAAATGTGATCAAACTTGTTTTAAGTTGGTCTGAAACAATCCAGAGTGTGTTATCATGAATTAGCATTTCTTTGTGTTCTGTCTGCCATGAGCACAAAGGTCACGGAGAAAATCAGCTTGAGTGAGTTGTTTTAAAATCAATGGCTCTATCAAAGATAAATGTTATACCTGCATATGTTGGAATGCTGATCTCAGCAATTCTGTTTTAACTGTTATTGAAGCTCAGCACTTTCCTTCAATGTGTATTTGCTTACATGGATATATATTTAGATAGCTCCTTTGAGAACTGCTTAAGAGCCGCGCGTCACTTTGTGCTTGACAAAAGCCAGGCTCTTCATTTCAGCAAGAGTTGGAACTTGTCCAGAATTGATAAGCGTAATTCAAATTCAAAATATGCATGACTTTTTTTTCAGATAAAAGTTAGGCAGCATCTCTTCTCCCTCAATCTCCTGAGAATCCTAAAAATAACGTCATAACCGTTTCAAATTACAGCCAACAGAAAGCAGGGAGAGGGTCTATGAGTAGAAGATATTTGCAAATCTGACATGGGGAGTAGAATATCATCAGTCTCCTAAATTAGAAAAAGTTGAAGCAATTTGGAACAGATTTTAAATCTCCAGTCAACGTAAGATCATACCTAGAACTCACTCTTATCTTTTATGTTACTATCTCCTTAAGAATTTCCCCCGCTCTTAGTATTTCCTGGTTACACTCTGAATTCACTTACCTGTAAATCTGTATTAGTGCTGGAGTGAGGATGAACAGAAGTAGGGGCCCATATTTTTCTTATACTTCGGATCTAGAACCTAGCTCATTTCAGAATTGTACACTCATGCATACTCATTTGATGTTTATTAGGAAGAGAATTTAACTCTTTGAATGTGTTGATAATATTTACTGTTTTTGTCTCATTATTAAAAGAAAAGGAATTGTGGAATTACTTGTAGATGTATTCTTCATAGTTTGAACATCCATTGTTATGAATAAGCAGCTACATTGCCATCCAGGAGAGTACAACAGTCTATAGAGAGGCAGACTATCCTGTGCATTTTGGTTTTGTGGCCTTTATATGTATTGACAAAGGTTAGATTTGTTGCATAATCACGCAGGTATTACATTTCACAACTGCAGTGCAGCTCTAAACATTTTCAATTTATTTTTAGCGAGCTCTTGGCTGTAATATTTCATTGCAGTCATGGCACCTAAGAAGAAGAACGTGAAGAAGAACAAAGCAGCAGATATCAGTGAAATGACTATCATTGTGGAAGATGGCCCC.
2.2 analysis of association between SNP sites of amplified fragment region sequence of KLHL31 gene and growth traits
And (3) carrying out association analysis on the 3 SNP loci and the growth traits.
As shown in Table 2, NC _052534.1: g.87828628G > C locus has significant correlation with weight at 91 days of age (p <0.05), wherein the weight at 91 days of GG homozygous genotype individuals is significantly larger than that of GC heterozygous genotype individuals (p <0.05), and CC homozygous genotype individuals have no significant difference with GG homozygous genotype individuals and GC heterozygous genotype individuals (p > 0.05); as shown in Table 3 and Table 4, NC-052534.1: g.87828969C > T locus has significant correlation (p <0.05) with weight at 77 days and weight at 84 days, wherein the weight at 77 days and the weight at 84 days of CC homozygous genotype individuals are both significantly larger than TT homozygous genotype (p <0.05), while the weight at CT heterozygous genotype individuals have no significant difference (p >0.05) with CC homozygous genotype individuals and TT homozygous genotype. The association of other SNP loci with growth traits did not reach a significant level (p > 0.05).
TABLE 2 NC-052534.1 g.87828628G > C site associated with growth trait
TABLE 3 NC-052534.1 g.87828969C > T site is associated with growth trait
TABLE 4 NC-052534.1 g.87828969C > T site associated with growth 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.
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gcttaagagc cgcgcgtcac tttgtgcttg acaaaagcca ggctcttcat ttcagcaaga 480
gttggaactt gtccagaatt gataagcgta attcaaattc aaaatatgca tgactttttt 540
ttcagataaa agttaggcag catctcttct ccctcaatct cctgagaatc ctaaaaataa 600
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gaagcaattt ggaacagatt ttaaatctcc agtcaacgta agatcatacc tagaactcac 780
tcttatcttt tatgttacta tctccttaag aatttccccc gctcttagta tttcctggtt 840
acactctgaa ttcacttacc tgtaaatctg tattagtgct ggagtgagga tgaacagaag 900
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Claims (5)
1. The application of a primer pair for detecting SNP molecular markers related to the weight of chickens in chicken genetic breeding is characterized in that the SNP molecular markers have two SNP loci of SNP1 and SNP2, the SNP1 is NC-052534.1: g.87828628G > C, the SNP2 is NC-052534.1: g.87828969C > T, and the genotype of the SNP1 comprises GG, GC and CC; the genotype of the SNP2 includes CC, CT, and TT; when the genotype of the SNP1 locus is GG, the weight of 91-day-old chickens is large; when the genotype of the SNP2 locus is CC, the weight of a chicken aged 77 days and the weight of a chicken aged 84 days are heavy, and the chicken is female Ningdu sanhuang chicken.
2. The use of claim 1, wherein the primer pair has the sequence shown in SEQ ID NO 2-3.
3. A method for identifying chicken growth traits by using a primer pair for detecting SNP molecular markers related to chicken body weight is characterized in that the SNP molecular markers have two SNP loci of SNP1 and SNP2, the SNP1 is NC-052534.1: g.87828628G > C, the SNP2 is NC-052534.1: g.87828969C > T, and the genotype of the SNP1 comprises GG, GC and CC; the genotype of the SNP2 includes CC, CT, and TT;
the method comprises the following steps:
(1) extracting the genome DNA of the chicken to be detected, and carrying out PCR amplification by using a primer pair to obtain an amplification product;
(2) sequencing the amplification product, detecting the genotype of the SNP1 locus, and when the genotype of the SNP1 locus is GG, weighing 91-day-old chickens; detecting the genotype of the SNP2 locus, wherein when the genotype of the SNP2 locus is CC, the weight of the chicken at the age of 77 days and the body weight of the chicken at the age of 84 days are determined;
the chicken is a female Ningdu sanhuang chicken, and the sequence of the primer pair is shown as SEQ ID NO. 2-3.
4. The method according to claim 3, wherein in step (1), the PCR amplification system is: template DNA 2. mu.L, 2 × Rapid Taq Master Mix 25. mu.L, upstream primer 2. mu.L, downstream primer 2. mu. L, ddH 2 O 19μL。
5. The method of claim 4, wherein in step (1), the reaction procedure of the PCR amplification is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 15s, and 35 cycles; completely extending for 3min at 72 ℃; storing at 4 ℃.
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