CN114854867B - Molecular marker related to abdominal fat character of yellow-feathered broiler chicken and application thereof - Google Patents
Molecular marker related to abdominal fat character of yellow-feathered broiler chicken and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker related to the 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 in a promoter NC-006088.5 g.11372123G>And (A). The invention discovers thatCD36The abdominal fat character of the yellow-feathered broilers is influenced by the mutation of the gene promoter region. Therefore, the molecular marker can be applied to breeding individuals with less abdominal fat deposition, and comprises the following steps: s1, extracting DNA of yellow-feathered broilers to be detected; s2, obtaining and sequencing a target fragment: amplifying the DNA of the chicken to be detected by PCR by using a forward primer pair and a reverse primer pair, sequencing the obtained product, and analyzing sequence bases; and S3, selecting and reserving individuals judged as AA genotypes during breeding according to the analysis result. The molecular marker is beneficial to breeding dominant homozygous genotype yellow-feathered broilers, and reduces the 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
The chicken is the second largest meat product in meat consumption in China, and is second to the pork. The yellow-feather broilers are completely and autonomously cultivated varieties in China, and compared with large white-feather broilers, the yellow-feather broilers are high in disease resistance and delicious in meat quality, and are widely popular with consumers. In recent years, yellow-feathered broilers have been greatly developed in the aspects of daily gain, growth speed, feed conversion rate and the like, but in commercial groups, along with improvement of production performance, the body fat content is too high, particularly the abdominal fat deposition is serious, and other negative effects also appear, so that great economic loss is brought to the broilers. Because of excessive deposition of fat at the belly of yellow-feathered broilers and the like, the redundant fat can only be discarded as waste, thereby not only reducing the feed conversion rate, but also influencing the meat quality and reducing the dressing percentage and the economic benefit. In addition, as the standard of living of people increases, consumers are more pursuing healthy, low-fat meat products. Therefore, fat deposition of yellow-feathered broilers is an urgent problem to be solved, and the problem of fat deposition in the abdomen is the most serious and urgent problem.
CD36 is a transmembrane protein, also known as Fatty Acid Translocase (FAT), located on chromosome 1 of chickens (reference genome GRCg6a, chrome 1). It has a fatty acid binding domain in its structure, is mainly involved in the uptake of fatty acids, and plays an important role in various metabolic diseases, including atherosclerosis, NAFLD, diabetes, and the like. It has been found that CD36 selectively transports linoleic acid in chicken muscle tissue, resulting in higher linoleic acid deposition in chicken muscle tissue. The gene polymorphism research of CD36 lays a foundation for uncovering the mechanism of abdominal fat deposition.
The Single Nucleotide Polymorphism (SNP) molecular marker has the heritable characteristic and can be widely used for large-group large-scale breeding and screening of yellow-feathered broilers.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a molecular marker related to the abdominal fat character of yellow-feathered broilers and application thereof, and the invention discovers that the mutation of a CD36 gene influences the abdominal fat character of the yellow-feathered broilers by a molecular marker method, wherein the CD36 gene is a known gene sequence and is positioned on a No. 1 chromosome (reference genome GRCg6a, chrome 1) of a chicken. 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.
Another object of the present invention is to provide the use of the above molecular markers in individuals with a high elimination of chicken abdominal fat deposits.
Another object of the present invention is to provide a method for eliminating individuals with severe chicken abdominal fat deposition by using the above molecular markers.
In order to achieve the purpose, the invention is realized by the following scheme:
a method for detecting a molecular marker related to the abdominal fat trait of yellow-feathered broilers is disclosed, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the mutation of the molecular marker is positioned at a promoter NC-006088.5 g.11372123G > -A. The invention finds that in the detected yellow-feathered broiler group, the gene mutation type AA abdominal FAT deposition is less, so the research indicates that the mutation of Fatty Acid Transposase (FAT) in chicken chromosome I, namely CD36 and CD36 promoter region can cause the change of the transcription efficiency, and further influence the fatty acid uptake. Individuals with less abdominal fat deposition can therefore be obtained by screening for AA types.
According to the method for molecular marking, the specific steps of the method for molecular marking are as follows:
s1, designing a forward primer pair and a reverse primer pair according to a chicken CD36 gene sequence to obtain an amplification primer:
forward primer F (SEQ ID NO: 2): 5' TGCCAGGATTGCTGTTGGAA-3
Reverse primer R (SEQ ID NO: 3): 5' CGGTGTTCCTGCCAACTACTACTACT-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 isolated product obtained in the step S3 to obtain a favorable genotype of the chicken abdominal fat trait, wherein the mutation of the obtained molecular marker is positioned at a promoter NC-006088.5 g.11372123G >A, the favorable genotype is an individual of AA genotype, and the chicken abdominal fat trait comprises the abdominal fat weight and abdominal fat rate of chicken.
Therefore, the invention also claims the application of the primer for detecting the molecular marker in eliminating individuals with serious chicken abdominal fat deposition.
Further, the reaction conditions of the PCR amplification in step S2 are: pre-denaturation at 94 ℃ for 2min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 40s, and 34 cycles; extension at 72 ℃ for 5min.
Further, the reaction system of the PCR amplification in step S2 is as follows:
further defined, the electrophoretic separation described in step S3 is performed using agarose gel at a concentration of 1.5%.
Further defined, the CD36 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 result of agarose gel electrophoresis.
FIG. 2 shows the different genotypes of the polymorphic sites of the CD36 gene.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present 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 specific 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 character determination.
409 Jiangfeng spotted-brown chickens of 100 days old are randomly selected, 1.2mL of infrawing venous blood is collected by using a vacuum anticoagulation tube (heparin is used as a built-in anticoagulant), and the collected blood is stored in a low-temperature refrigerator at minus 80 ℃ for subsequent DNA extraction. The total bore weight and abdominal fat weight after slaughter were measured and abdominal fat rate was calculated (the PRC agricultural industry Standard NY/T823-2004).
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 apparatus (Bio-Rad, USA)
4. And (4) preparing a buffer solution and a common reagent.
The DNA extraction reagent adopts NRBC Blood DNA Kit
5. Design and Synthesis of primers
Based on the reported sequence of the chicken CD36 Gene (Gene ID: 417730), primer design was performed using NCBI Primer-BLAST, which was synthesized by Guangzhou division, beijing Enginko Biotechnology, inc.
Example 1
1. Extraction of genomic DNA:
all DNA extraction operations of chicken individuals to be detected are carried out according to the NRBC Blood DNA Kit (OMEGA, D0715) instruction, and the extracted DNA is stored in a low-temperature refrigerator at-20 ℃ after concentration and purity detection for subsequent PCR amplification.
2. Obtaining a target fragment of the chicken CD36 gene:
the reference genome was GRCg6a, chrome 1 by following the reported sequence of the chicken CD36 Gene (Gene ID: 417730).
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-CD36：5’-TGCCAGGATTGCTGTTGGAA-3’；
R-CD36：5’-CGGTGTTCCTGCCAACTACT-3’。
the primers are adopted to carry out PCR amplification (the amplification length is 1150 bp) on a sample to be detected
The following solutions or reagents were included in 30ul of the reaction system:
TABLE 1 PCR reaction mix 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 2min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 40s, and 34 cycles; extension at 72 ℃ for 5min.
4. After the reaction is finished, taking the PCR reaction solution to perform agarose gel electrophoresis on the amplification product, detecting the PCR product, and judging the result according to the size of the product:
and detecting the PCR amplification product by using 1.5% agarose gel electrophoresis containing EB under the conditions of 100V voltage and 20min time, observing a band (1150 bp, shown in figure 1) in a gel imaging system, recovering and purifying, sequencing and analyzing sequence bases.
The detection finds that NC _006088.5 g.11372123G >has missense mutation (as shown in FIG. 2).
Example 2
The invention is used for detecting and analyzing the correlation between different genotypes of the polymorphic site of the CD36 gene and the abdominal fat trait of yellow-feathered broilers.
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 SNP site 11372123G >. Among them, AA genotype individuals showed the lowest abdominal fat weight and abdominal fat rate, and GG genotype individuals showed the highest abdominal fat weight and abdominal fat rate, and the specific information is shown in the following table.
TABLE 2 correlation analysis of different genotypes of CD36 g.11372123G >
The table shows that the abdominal fat weight (P < 0.001) and the abdominal fat rate (P < 0.002) of the yellow-feathered broilers reach extremely remarkable levels by the 3 genotypes. The abdominal fat weight and abdominal fat rate of AA genotype individuals are obviously lower than those of GG genotype individuals, and the AA genotype individuals are dominant genotypes.
Therefore, individuals with less abdominal fat deposition can be bred through the CD36 gene marker-assisted selection, 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> south China university of agriculture
<120> molecular marker related to abdominal fat character of yellow-feathered broilers and application thereof
<130> 2021.12.01
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1150
<212> DNA
<213> spotted-brown chicken (chicken)
<400> 1
tgccaggatt gctgttggaa ggccataaat taatctatac aatggttttg gcctgttata 60
aggagatttg tacagatcac tgagtgtaca tacgctgttt aggtacagat agattacagt 120
tgtagacatt aaagcggctg gaagaaacac agtagataaa ctgactgata tcaactgggt 180
gctgtctgta gtgtttgctc aagattctgg catgctccca tgtgtacttg gactttggaa 240
cagaaaatgt ttggtaatac ctaggttgga taaatgttgt aaaaactgtt ctcacacgcc 300
aggaattaca gtgggatgga atttcttgtt gcactaaggt gcagtgttgt gatgggaatt 360
gcagtctgct cacttgagga gtacagggta ctgttagtga tatggtgaag atttatccct 420
aaagaggaag tgggcagcag cctaacagca aggtttaatt acggaagatg aagtctgcat 480
taaatagaac ctggcagctg agtaaaggaa tttggaaggc aagaccggtg aaaacctaac 540
tagaaagcca ctaaccaaga agggtgcgca gtggaagaaa tagctaccag aagctgaagg 600
aaaagagtat ttatgtgtct gtaatcctgt ggctgagatt acccagatag gcttgggagt 660
gccctgctga atcctctcca gcaccattta ggaaatctga taagtggctg ttgttacttt 720
ttccccaaaa cagagaagcc tgggtgtaat tagatggagg gtgtgtactc caccgcagtg 780
cctggggttc ttcatgctgt gccctgtcct ctcaaagcaa aaggcagtgg tgaaggacac 840
agcctattac ctttctagct gttgtgggat ggaagggcac tgcactgctc ttagcagtcc 900
aacatttcta ctcctgcact gagtctgact ctgcatatag agaagtctgt gctgctcctg 960
gcttcttctc agttcctctc tcagttctgg aacaggtaag tggaggatga ggattgcctt 1020
tccttactta gcacatctgc tctgtgtatg ggcaaagctg ttgaagattc attttcttgg 1080
gagtaggagc agttcaaaaa gtgtcctgaa tgacagctgg atggggaaaa agtagttggc 1140
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgccaggatt gctgttggaa 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cggtgttcct gccaactact 20
Claims (6)
1. A method for detecting a molecular marker related to the abdominal fat character of yellow-feathered broilers is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the mutation of the molecular marker is positioned at a promoter NC-006088.5 g.11372123G > A thereof;
the method comprises the following specific steps:
s1, designing a forward primer pair and a reverse primer pair according to a chicken CD36 gene sequence to obtain an amplification primer:
the forward primer F is SEQ ID NO:2:5' TGCCAGGATTGCTGTTGGAA-3
The reverse primer R is SEQ ID NO:3:5' CGGTGTTCCTGCCAACTACTACTACT-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 isolated product obtained in the step S3 to obtain a favorable genotype of the chicken abdominal fat character, wherein the mutation of the obtained molecular marker is located at a promoter NC _006088.5 g.11372123G > -A, the favorable genotype is an individual of an AA genotype, and the chicken abdominal fat character comprises the abdominal fat weight and the abdominal fat rate of the chicken.
2. The application of the primer of claim 1 in individuals with severe yellow feather broiler abdominal fat deposition.
3. The method for detecting the molecular marker related to the abdominal fat trait of yellow-feathered broilers according to claim 1, wherein the reaction conditions of the PCR amplification in the step S2 are as follows: pre-denaturation at 94 ℃ for 2min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 40s, and 34 cycles; extension at 72 ℃ for 5min.
4. The method for detecting the molecular marker related to the abdominal fat trait of yellow-feathered broilers according to claim 1, wherein the reaction system for PCR amplification in the step S2 is as follows:
5. the method for detecting the molecular marker related to the abdominal fat trait of yellow-feathered broilers, as claimed in claim 1, wherein the electrophoretic separation in the step S3 is performed by agarose gel separation, and the concentration of the agarose gel is 1.5%.
6. The method for detecting the molecular marker related to the abdominal fat trait of yellow-feathered broilers according to claim 1, wherein the CD36 gene sequence in the step S1 is positioned in a fatty acid binding region existing on a chicken No. 1 chromosome structure.
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Non-Patent Citations (2)
| Title |
|---|
| FAT/CD36融合蛋白的表达及其对鸡腹脂沉积的特异性调控;束刚等;《中国农业科学》;20090210;第42卷(第02期);全文 * |
| 黄羽肉鸡FAT/CD36 cDNA的分子克隆及其发育性表达;冯嘉颖等;《中国农业科学》;20071010;第40卷(第10期);全文 * |
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