CN114790477A - Molecular marker related to abdominal fat character of yellow-feathered broilers and application thereof - Google Patents
Molecular marker related to abdominal fat character of yellow-feathered broilers and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker related to abdominal fat character of yellow-feathered broilers and application thereof. The nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1, and the mutation of the molecular marker is positioned at the seventh exon g.8419598C > T. The invention discovers that the abdominal fat character of yellow-feathered broilers is influenced by the DKK3 gene promoter region mutation. Therefore, the molecular marking method can be applied to breeding individuals with less abdominal fat deposition, eliminating individuals with serious abdominal fat deposition, extracting DNA of yellow-feathered broilers to be detected, amplifying the DNA of the chickens to be detected by PCR through obtaining target fragments and sequencing by using forward and reverse primers, sequencing the obtained products, analyzing sequence bases, and selecting and reserving the individuals judged as the CT genotype during breeding according to the analysis result. The molecular marker is beneficial to breeding dominant heterozygous genotype CT type yellow-feathered broilers, and reduces abdominal fat deposition of the broilers.
Description
Technical Field
The invention relates to the technical field of poultry genetic breeding, in particular to a molecular marker related to the abdominal fat character of yellow-feathered broilers and application thereof.
Background
China is the first broiler to be slaughtered in the world, and the annual slaughtering amount exceeds more than 100 billions. With the improvement of chicken consumption of people, the requirements on chicken quality are also improved, the proper fat content can increase the flavor of meat, and the tenderness and juiciness are improved. However, excessive deposition of abdominal fat affects the quality of meat. The need of enterprises at present has also changed from a taste of pursuit of growth rate of broiler chickens to development of new low-fat lines, because the excessive pursuit of growth rate of broiler chickens in production can lead to excessive deposition of chicken abdominal fat and cause early expression of obesity genes. This not only results in a decrease in feed utilization, an increase in feeding costs, and a decrease in economic benefits, but also affects the health of chickens and the flavor of their meat, and at the same time, fat as a waste product of production can cause serious environmental pollution. A key gene DKK3 capable of regulating fat differentiation is found in upstream regulatory factors of Wnt signaling pathways in the previous period, and then, whether the gene influences the fat character of broiler chickens through the change of single nucleotide polymorphism is researched.
DKK3 (dickkopf 3) is a member of the DKK family of proteins, a secreted glycoprotein, and plays important regulatory roles in tissue development, homeostatic regulation, and various pathological states. The research of DKK3 focuses on tumors at present, and a plurality of researches report that DKK3 is applied to a plurality of human cancers as a tumor inhibitor and can inhibit the growth of tumor cells after DKK3 is over-expressed. With the intensive research, DKK3 was found to promote the growth of tumor cells in rectal cancer. The gene polymorphism of DKK3 is closely related to abdominal fat deposition in the present study.
The Single Nucleotide Polymorphism (SNP) molecular marker has heritable characteristics and can be widely used for large-scale breeding and screening of large groups of yellow-feathered broilers.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a molecular marker related to the abdominal fat character of yellow-feathered broilers and applies the molecular marker to marker-assisted selection of the abdominal fat character, and the invention discovers that the abdominal fat character of the yellow-feathered broilers is influenced by DKK3 gene mutation. Therefore, the molecular marker is beneficial to breeding dominant homozygous genotype yellow-feathered broilers, eliminating individuals with severe abdominal fat deposition and improving the production performance of the yellow-feathered broilers.
In order to achieve the purpose, the invention is realized by the following scheme:
a molecular marker related to the abdominal fat character of yellow-feathered broilers has a nucleotide sequence shown in SEQ ID NO. 1, and is positioned at the mutation of the seventh exon g.8419598C > T in the chicken DKK3 gene. In the detected yellow-feathered broiler group, the gene mutation type CT abdominal fat deposition is less, so that the change of the transcription efficiency of the DKK3 due to the mutation of the seventh exon region is obtained, and the fatty acid uptake is further influenced. Therefore, individuals with less abdominal fat deposition can be obtained by screening for CT type.
Further, a molecular marking method for eliminating individuals with serious chicken abdominal fat deposition comprises the following specific steps:
s1, designing a forward and reverse primer pair according to a chicken DKK3 gene sequence to obtain an amplification primer:
forward primer F (SEQ ID NO: 2): 5'-GGTCTTCTGTGGTGGACCC-3'
Reverse primer R (SEQ ID NO: 3): 5'-CTGAAATGGGTGACTGAGGTG-3'
S2, performing PCR amplification on chicken genome DNA by using the amplification primer obtained in the step S1 to obtain an amplification product, wherein the nucleotide sequence of the amplification product as a molecular marker is shown as SEQ ID NO. 1, sequencing the obtained product, and analyzing sequence bases;
s3, carrying out electrophoretic separation on the amplification product obtained in the step S2 to obtain a separation product;
s4, carrying out genotype analysis on the separated product obtained in the step S3 to obtain a favorable genotype of the chicken abdominal fat traits, wherein the mutation of the obtained molecular marker is positioned at the seventh exon g.8419598C > T of the favorable genotype, the favorable genotype is an individual of the CT genotype, and the chicken abdominal fat traits comprise the abdominal fat weight and the abdominal fat rate of the chicken.
Further, the reaction conditions of the PCR amplification in step S2 are: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 40s, for 34 cycles; extension at 72 ℃ for 5 min.
Further, the reaction system of the PCR amplification in step S2 is as follows:
DNA template 2. mu.L
F-DKK3 0.6μL
R-DKK3 0.6μL
2xTaq Master Mix 15μL
ddH2O 11.8μL。
Further, the electrophoresis separation described in step S3 is performed by agarose gel separation, and the concentration of the agarose gel is 1.5%.
Further, the DKK3 gene sequence described in step S1 is located in a fatty acid binding region present on the chicken chromosome 1 structure.
Compared with the prior art, the invention has the following beneficial effects:
1. the molecular marker provided by the invention is beneficial to breeding yellow-feather broilers with less abdominal fat deposition, eliminating individuals with serious abdominal fat deposition and improving the production performance of the yellow-feather broilers.
2. The molecular marker influencing the abdominal fat weight of the chicken is applied to the genetic breeding of the chicken, is more efficient and can be stably inherited.
Drawings
FIG. 1 shows the results of agarose gel electrophoresis.
FIG. 2 isDKK3The polymorphic sites of the gene are of different genotypes.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
The reagents, instruments, experiments, etc. involved in the present invention are described as follows:
1. experimental animals and trait determination.
451 Jiangfeng spotted-brown chickens of 100 days old are randomly selected, are not grouped, and are collected into 1.2 mL of blood of the veins under the wings by using a vacuum anticoagulation tube (heparin is used as a built-in anticoagulant), and are stored in a low-temperature refrigerator at-80 ℃ for subsequent DNA extraction. The total bore weight and abdominal fat weight after slaughter were measured, and abdominal fat ratio was calculated (agricultural industry standard NY/T823-2004 of China's republic of China).
2. Drugs and enzymes.
DNA Marker, Ethidium Bromide (EB), Shanghai Biotechnology engineering Co., Ltd.
Taq enzyme, ddH2O, Beijing kang, century Biotechnology Ltd
3. The main equipment is equipment.
MJ Mini gradient PCR instrument (Bio-Rad, USA)
4. And (4) preparing a buffer solution and a common reagent.
None of the DNA extraction reagents are from NRBC Blood DNA Kit
5. Design and Synthesis of primers
Based on the reported sequence of the chicken DKK3 Gene (Gene ID: 396023), NCBI Primer-BLAST was used for Primer design, which was synthesized by Guangzhou division, Beijing Ongchong Biotechnology, Inc.
Example 1
1. Extraction of genomic DNA:
all DNA extraction operations of chicken individuals to be detected refer to a conventional Kit DNA extraction method, the DNA extraction is carried out according to the NRBC Blood DNA Kit (OMEGA, D0715) instruction, and the extracted DNA is stored in a low-temperature refrigerator at the temperature of-20 ℃ after being subjected to concentration and purity detection and is used for subsequent PCR amplification.
2. Chicken with egg yolkDKK3Obtaining the target fragment of the gene:
by means of the reported chickensDKK3Gene sequence (Gene ID: 396023).
3. Performing PCR amplification by using the extracted chicken DNA as a template:
primer design was performed using NCBI Primer-BLAST, which was synthesized by Guangzhou division, Beijing Ongkogaku Biotechnology Ltd;
the primer sequences are as follows:
F-DKK3：5’- GGTCTTCTGTGGTGGACCC -3’；
R-DKK3：5’- CTGAAATGGGTGACTGAGGTG -3’。
the primers are adopted to carry out PCR amplification (the amplification length is 900 bp) on a sample to be detected
The following solutions or reagents were included in a 30ul reaction system:
PCR reaction mixing system
| Mixed sample | Dosage (mu L) |
| DNA template | 2 |
| F-CD36 | 0.6 |
| R-CD36 | 0.6 |
| 2xTaq MasterMix | 15 |
| ddH 2 O | 11.8 |
| Total volume | 30 |
The above solutions were mixed and subjected to PCR reaction under the following conditions: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 40s, for 34 cycles; extension at 72 ℃ for 5 min.
4. After the reaction is finished, taking the PCR reaction solution to carry out agarose gel electrophoresis on the amplification product, detecting the PCR product, and judging the result according to the size of the product:
and (3) detecting the PCR amplification product by using 1.5% agarose gel electrophoresis containing EB under the voltage of 100V for 20min, observing a band (900 bp, as shown in figure 1) in a gel imaging system, recovering and purifying, sequencing, and analyzing sequence bases.
Detection revealed GTACTCGTACTC that a missense mutation was present (as shown in FIG. 2).
Example 2
The invention is rightDKK3The detection application of the correlation analysis of different genotypes of the gene polymorphic sites and the abdominal fat character of yellow-feathered broilers is carried out.
Performing association analysis of SNP sites and abdominal fat traits (abdominal fat weight and abdominal fat rate) by using SAS 9.0 software, wherein the adopted model is a Proc-GLMR function model, and the model formula is as follows:
Y= u + F + M + S + G + e
wherein Y is a phenotypic value, u is a population mean, F is a paternal effect, M is a maternal effect, S is a gender effect, G is a genotype effect, and e is a random residual.
And displaying a correlation analysis result: the association of the SNP site 8419598C > T with the abdominal fat weight trait reaches a very significant level (P = 0.0003), and is very significantly related to the abdominal fat weight trait (P = 0.0004). Among them, the CT genotype individuals showed the lowest abdominal fat weight and abdominal fat rate, and the CC genotype individuals showed the highest abdominal fat weight and abdominal fat rate, and the specific information is shown in the following table.
Table 2. DKK3 g.8419598 C>Correlation analysis of T different genotypes and growth and development traits
The results show that the abdominal fat weights (P < 0.001) and abdominal fat rates (P < 0.001) of the yellow-feathered broilers reach extremely significant levels by the 3 genotypes. The abdominal fat weight and abdominal fat rate of CT genotype individuals are obviously lower than those of CC type individuals, so that the CT genotype commercial generation chickens with heterosis can be cultivated.
Thus, can passDKK3The gene marker assisted selection is adopted to select individuals with less abdominal fat deposition, and the production performance of yellow-feathered broilers is improved.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> molecular marker related to abdominal fat character of yellow-feathered broilers and application thereof
<141> 2022-05-06
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 900
<212> DNA
<213> Artificial sequence (SEQ ID NO:1)
<400> 1
gctttgctat taggaacttg gttctcactc tgtaagtaga aaactctcaa ctttccagct 60
gtgaagaagc aatcagatgt ttacagggtc ttctgtggtg gacccgacaa ttttatatag 120
ctagtatgct tttacagggg caggatatga tgaattttat taagaactag caagaataac 180
gtgagatgta ctgtatgacc agacgtcttt ctttttccag cagccacagt actacatctg 240
tgtgtgaact gtcctccaat gaaaccagga aaaacgaaaa agaagatccc ttgaacatgg 300
atgagatgcc atttatcagt ttaataccca gagatattct ttctgattac gaagaaagca 360
gcgtcattca ggaagtgcgt aaagaattag aaagcctgga ggaccaagca ggtgtgaagt 420
ctgagcatga cccggctcat gacctatttc tgggagatga aatatgaagt tcaaacacca 480
gtttagttag tcctagaaat tgttgtctag tgtcttgctt acatacaccc ttaacagata 540
ctgctggata gaagtgcaat aaacatcttc attgagcatc cgttttcgtg caccaaacct 600
gcatgttcaa attcatgttg aattcactca atctttggac caaactttcc atcaaagaca 660
aatgagaaag gcatcagtgt ttcctttgga ttaatccttt cctttgtaca gcagaaataa 720
acgtatcagt actcgtactc attaaaaaaa cacacggagc atacataatc tcaagttatt 780
agtgactaca gaaacaactg ctaagtattt cctgtacaca cacttgaagt tatttttctg 840
gagtaagaaa tttaatacca ctttaatacc acctcagtca cccatttcag caattttgga 900
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggtcttctgt ggtggaccc 19
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctgaaatggg tgactgaggt g 21
Claims (7)
1. The molecular marker related to the abdominal fat trait of yellow-feathered broilers is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the molecular marker is positioned at the mutation position of the seventh exon g.8419598C > T in the chicken DKK3 gene.
2. The molecular marker related to abdominal fat trait of yellow-feathered broilers, as claimed in claim 1, wherein the molecular marker is applied to individuals with severe abdominal fat deposition.
3. The molecular marking method for individuals with severe chicken abdominal fat deposition as claimed in claim 2, wherein the molecular marking method comprises the following steps:
s1, designing a forward and reverse primer pair according to a chicken DKK3 gene sequence to obtain an amplification primer:
forward primer F (SEQ ID NO: 2): 5'-GGTCTTCTGTGGTGGACCC-3'
Reverse primer R (SEQ ID NO: 3): 5'-CTGAAATGGGTGACTGAGGTG-3'
S2, performing PCR amplification on chicken genome DNA by using the amplification primer obtained in the step S1 to obtain an amplification product, wherein the nucleotide sequence of the amplification product as a molecular marker is shown as SEQ ID NO. 1, sequencing the obtained product, and analyzing sequence bases;
s3, carrying out electrophoretic separation on the amplification product obtained in the step S2 to obtain a separation product;
s4, carrying out genotype analysis on the separated product obtained in the step S3 to obtain a favorable genotype of the chicken abdominal fat traits, wherein the favorable genotype is an individual of the CT genotype, and the mutation for obtaining the molecular marker is positioned at the seventh exon g.8419598C > T of the favorable genotype, and the chicken abdominal fat traits comprise the abdominal fat weight and the abdominal fat rate of the chicken.
4. The molecular labeling method of claim 3, wherein the reaction conditions of the PCR amplification in step S2 are: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 40s, for 34 cycles; extension at 72 ℃ for 5 min.
5. The molecular labeling method according to claim 3, wherein the reaction system for PCR amplification in step S2 is as follows:
DNA template 2. mu.L
F-DKK3 0.6μL
R-DKK3 0.6μL
2xTaq Master Mix 15μL
ddH2O 11.8μL。
6. The molecular labeling method of claim 3, wherein the electrophoresis separation in step S3 is performed by agarose gel separation, and the concentration of the agarose gel is 1.5%.
7. The method according to claim 3, wherein the DKK3 gene sequence of step S1 is located at a fatty acid binding region present on a chicken chromosome 1 structure.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116042846A (en) * | 2022-09-30 | 2023-05-02 | 华南农业大学 | Molecular marker related to slaughter performance of yellow-feather broilers and application thereof |
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Application publication date: 20220726 |