CN116042841B - Molecular marker related to leg hair follicle size of yellow-feather broiler and application thereof - Google Patents
Molecular marker related to leg hair follicle size of yellow-feather broiler and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker related to the size of a shank hair follicle of yellow-feather broiler and application thereof, 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 g.320C > A of a 4 th intron region of the molecular marker. The invention discovers that the mutation of the intron region of the HOXB3 gene affects the size of the leg hair follicle of the yellow-feather broiler, provides a molecular marker related to the size of the leg hair follicle of the yellow-feather broiler, can be used for early selection in family breeding, and can also provide a molecular basis in selection and matching. The SNP molecular marker provides a new method for screening the size and the shape of the skin hair follicles of the drumsticks, is favorable for breeding dominant genotype yellow-feather broilers, improves the size of the leg hair follicles of the chickens, can save production cost and accelerate genetic development, better serves the breeding of high-quality broilers, and has great economic value and scientific research value.
Description
Technical Field
The invention relates to the technical field of poultry genetic breeding, in particular to a molecular marker related to the size of a shank hair follicle of a yellow-feather broiler and application thereof.
Background
China is a large country for producing and consuming broilers, chicken is the second most popular in the whole meat consumer market in China, and is meat food and consumer products inferior to pork. In recent years, african swine fever frequently occurs in China, the consumption of pork by consumers is reduced, and the consumption of chicken is continuously increased. And because of the influence of the COVID-19, domestic animal living body trading market is shrinking, the consumption mode is gradually changed from live poultry trading to centralized slaughtering and ice fresh marketing, the first impression of consumers on the broiler chicken is changed from live chicken appearance characters such as feather color, feather body degree and the like to carcass characters such as carcass body type, skin color, hair follicle density and the like, and the breeding direction of the broiler chicken is also changed along with the change of the attention target. It has been found by investigation that the hair follicle is dense and thin, the skin compactness is good, and the bright yellow iced fresh chicken is favored by consumers.
The homeobox gene (HOX) is a kind of evolutionarily highly conserved homeodomain transcription factor coding gene, the HOX gene of vertebrates has four clusters on chromosome, and its sequence contains a conserved sequence formed from 180-183 bases, and the polypeptide region formed from amino acids translated by said sequence is called homeodomain. HOXB3 (homeobox B3) belongs to the HOX gene family member gene and is located on chromosome 27 of chicken (reference genome GRCg6a, chrome 27). There have been studies to find that almost all other members are expressed in the skin or hair follicle, except for a very small number of HOX gene family members (HOXA 13, HOXD1 and HOXD 12). The transcription factor coded by the HOX gene plays an important role in the morphogenesis and periodical change process of hair follicles, and can activate dormant mesenchymal cells through a Wnt signal path so as to promote regeneration of hair follicles, and abnormal expression of Wnt can influence the development process of the feather buds in the feather bud formation process. As a molecular breeding means for livestock and poultry, a single nucleotide polymorphism (Single nucleotide polymorphism, SNP) molecular marker can be used for breeding yellow-feather broilers with good quality by researching the gene polymorphism of HOXB 3.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a molecular marker related to the size of the leg hair follicle of yellow-feather broiler and application thereof, and the invention discovers that the HOXB3 gene mutation affects the size of the leg hair follicle of yellow-feather broiler, so that the molecular marker is beneficial to breeding dominant genotype yellow-feather broiler, eliminating individuals with larger leg hair follicles and reducing the size of the leg hair follicle of yellow-feather broiler.
It is another object of the present invention to provide the use of the above molecular markers for the removal of large individuals with leg hair follicles.
Another object of the present invention is to provide a method for rejecting larger individuals of leg hair follicles using the above molecular markers.
In order to achieve the above object, the present invention is realized by the following means: a molecular marker related to the size of the leg hair follicle of yellow-feather broiler is provided, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the mutation of the molecular marker is positioned at an intron G.320C > A.
Further, a molecular marker related to the size of the leg hair follicle of the yellow-feather broiler is provided, and the molecular marker is applied to individuals with bigger leg hair follicles.
Further, the specific steps of the molecular marking method are as follows:
s1, designing a forward and reverse primer pair according to the HOXB3 gene sequence of the chicken to obtain an amplification primer:
forward primer F (SEQ ID NO: 2): 5'-AAACAGGCCAAAGGAAGCAC-3'
Reverse primer R (SEQ ID NO: 3): 5'-CAGTATCTGCATCCTCGCTGT-3'
S2, carrying out PCR amplification on chicken genome DNA by using the amplification primer obtained in the step S1, obtaining an amplification product, wherein the nucleotide sequence of the amplification product serving 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 affecting the size of chicken leg hair follicles, wherein the mutation of the obtained molecular marker is located at an intron g.320C > A of the favorable genotype, the favorable genotype is an AA genotype individual, and the character is the size of chicken leg hair follicles.
Further, the reaction conditions for the PCR amplification in step S2 are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s; annealing at 57 ℃ for 15s; extending at 72 ℃ for 90s;34 cycles; extending at 72℃for 5min.
Further, the reaction system of the PCR amplification in the step S2 is as follows:
Green Taq Mix 15μL
DNA 1μL
F- HOXB3 1.2μL
R- HOXB3 1.2μL
ddH2O 11.6μL。
further, the electrophoresis separation in step S3 uses agarose gel separation, and the concentration of the agarose gel is 1.5%.
Further, the HOXB3 gene sequence described in step S1 is located in the chicken chromosome 27 structural region.
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 smaller leg hair follicles, removing individuals with larger leg hair follicles, and reducing the size of the leg hair follicles of the yellow-feather broilers.
2. The molecular marker affecting the size of the drumstick hair follicle can be applied to the genetic breeding of chickens, and is more efficient and can be inherited stably.
Drawings
For ease of illustration, the invention is described in detail by the following preferred embodiments and the accompanying drawings.
FIG. 1 shows agarose gel electrophoresis results.
FIG. 2 shows the polymorphic loci of the HOXB3 gene of different genotypes.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The invention will be further illustrated with reference to specific examples, but the invention is not limited to the examples.
The reagents, instruments, experiments and the like related to the invention are described as follows:
1. experimental animals and trait determination.
(1) The experimental group is provided by the south Wen poultry breeding limited company of Guangdong, 400 mixed family chickens (half of the male and female) are added for the 16 th generation of the N204 strain of the spotted-brown chickens, and the experimental group consists of 63 families, wherein 360 breeding time is 87 days old, and the rest 40 are bred to 138 days old, and the experimental group is used for comparative analysis of the hair follicle sizes among different breeding days old of the same breed, and finally genotype and phenotype characteristics of 332 individuals are determined.
(2) Leg hair follicle size (Feather follicle size on legs, LFFS): the relative area of hair follicles on the left shank of the chicken is measured by using an Image J software, the number of the hair follicles is not less than 5, the average value of the sizes of a plurality of hair follicles is the measured value of the individual, the same individual is measured for 2 times, the relative deviation between the 2 times is not more than 10 percent, the average value of the measured values of the two times is calculated to be the relative size of the hair follicles of the legs of the individual, and for the convenience of calculation, the measured value unit is converted into a kilopixel point (kpx) from a pixel point (px).
2. Drugs and enzymes.
Dnagarer, ethidium Bromide (EB), shanghai bioengineering, inc.
Taq enzyme, ddH2O, nanjinouzan Biotechnology Co., ltd
3. Main instrument equipment.
PC35 skin microscope (Germany CK company)
MJ Mini gradient PCR instrument (Bio-Rad, USA)
4. Buffer solution and common reagent are prepared.
No DNA extraction reagent comes from the kit
5. Primer design and Synthesis
Based on the reported chicken HOXB3 gene sequence (GeneID: ENSGALG 00000043219), a primer design was performed using NCBIPrimer-BLAST, which was synthesized by Guangzhou division, inc. of Biotechnology, beijing.
Examples
1. Extraction of genomic DNA:
all the DNA extraction operations of the chicken individuals to be detected are carried out according to NRBC Blood DNA Kit (OMEGA, D0715) instructions by referring to the conventional kit DNA extraction method, and the extracted DNA is stored in a-20 ℃ low-temperature refrigerator for subsequent PCR amplification after concentration and purity detection.
2. Obtaining target fragment of chicken HOXB3 gene:
the reference genome was GRCg6a by following the reported chicken HOXB3 gene sequence (GeneID: ENSGALG 00000043219).
3. Performing PCR amplification by taking the extracted chicken DNA as a template:
primer design was performed using NCBI Primer-BLAST, which was synthesized by Guangzhou division, inc. of Beijing qingke biotechnology Co., ltd;
the primer sequences were as follows:
F-HOXB3：5’- AAACAGGCCAAAGGAAGCAC -3’；
R-HOXB3：5’- CAGTATCTGCATCCTCGCTGT -3’。
PCR amplification (amplification length 1441 bp) is carried out on the sample to be detected by adopting the primer
The following solutions or reagents were contained in a 30ul reaction system:
TABLE 1 PCR reaction mixture
| Reagent(s) | Volume of |
| Green Taq Mix | 15μL |
| DNA | 1μL |
| F- HOXB3 | 1.2μL |
| R- HOXB3 | 1.2μL |
| ddH 2 O | 11.6μL |
The above solutions were mixed and PCR reactions were performed under the following conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s; annealing at 57 ℃ for 15s; extending at 72 ℃ for 90s;34 cycles; extending at 72℃for 5min.
4. After the reaction is finished, taking PCR reaction liquid, carrying out agarose gel electrophoresis on amplified products, detecting the PCR products, and judging the result according to the size of the products:
PCR amplified products were detected by EB-containing 1.5% agarose gel electrophoresis at a voltage of 100V for 20min, bands (1441 bp, as shown in FIG. 1) were observed in a gel imaging system, recovered and purified, and finally sequenced to analyze the sequence bases.
g.320C > A missense mutation was found to exist (as shown in FIG. 2).
Description of the preferred embodiments
The invention carries out the detection application of the correlation analysis of the different genotypes of the HOXB3 gene polymorphic locus and the size of the leg hair follicle of the yellow-feather broiler.
The correlation analysis of SNP loci and the size of the hair follicles on the legs is carried out by using SPSS 26.0 software, the adopted model is a mixed linear model, and the model formula is as follows:
Y ijklm =µ+G i +S j +H k +f l +e ijklm
wherein Y is a phenotype value, u is a population mean value, G i For genotypic effect, S j For sex effect, H k For hatching effect, f l Is family effect, e is random error.
And (3) displaying the correlation analysis results: the association of SNP locus g.320c > a with leg hair follicle size reached a significant level (P < 0.05). Of 332 individuals detected at this time, the CC genotypes are 58, the CA genotypes are 162 and the AA genotypes are 112, wherein the AA genotype individuals show smaller leg hair follicles, and the CC genotypes individuals show larger leg hair follicles, and specific information is shown in the following table:
TABLE 2 correlation analysis of HOXB3 g.320C > A different genotypes with the size and shape of the leg hair follicle
Note that: * Is significantly correlated at the level of 0.05; different letters indicate significant differences.
From the above table, the sizes of the leg skin hair follicles of 3 genotypes to yellow-feather broilers (P < 0.05) reach a significant level, the sizes of the leg skin hair follicles of CC genotype individual chickens are similar to those of CA type, and the sizes of the leg skin hair follicles of AA genotype individual chickens are significantly lower than those of CC type and CA type, so that the AA genotype is a dominant genotype. In breeding screening, CC and CA types are removed, only AA type is selected, and the genotype can be fixed in the population. The SNP locus can be used as auxiliary selection of the size and shape of the skin hair follicle of the drumstick part and molecular genetic breeding markers. Therefore, by auxiliary selection of HOXB3 gene markers, individuals with smaller leg hair follicles are bred, and the production performance of yellow-feather broilers is improved.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (6)
1. A method for detecting molecular markers related to the size of leg hair follicles of yellow-feather broilers is characterized in that the nucleotide sequence of the molecular markers is shown as SEQ ID NO. 1, and the mutation of the molecular markers is positioned at g.320C > A;
the method comprises the following specific steps:
s1, designing a forward and reverse primer pair according to the HOXB3 gene sequence of the chicken to obtain an amplification primer:
forward primer F (SEQ ID NO: 2): 5'-AAACAGGCCAAAGGAAGCAC-3' reverse primer R (SEQ ID NO: 3): 5'-CAGTATCTGCATCCTCGCTGT-3'
S2, carrying out PCR amplification on chicken genome DNA by using the amplification primer obtained in the step S1, obtaining an amplification product, wherein the nucleotide sequence of the amplification product serving 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 affecting the size of chicken leg hair follicles, wherein the mutation of the obtained molecular marker is positioned at g.320C > A of the favorable genotype, and the favorable genotype is an AA genotype individual, and has the characteristic of smaller chicken leg hair follicles.
2. Use of the method of claim 1 for eliminating individuals with large shank follicles in yellow-feathered broilers, the advantageous genotype being the AA genotype and the trait being smaller shank follicles.
3. The method according to claim 1, wherein the reaction conditions for the PCR amplification in step S2 are: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s; annealing at 57 ℃ for 15s; extending at 72 ℃ for 90s;34 cycles; extending at 72℃for 5min.
4. The method according to claim 1, wherein the reaction system for PCR amplification in step S2 is as follows:
5. the method according to claim 1, wherein the electrophoresis separation in step S3 is performed by agarose gel having a concentration of 1.5%.
6. The method according to claim 1, wherein the HOXB3 gene sequence of step S1 is located in the chicken chromosome 27 structural region.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102094022A (en) * | 2010-08-31 | 2011-06-15 | 李宁 | Gene Hoxc8 related to tassel head characters of chicken and application thereof |
| CN103898102A (en) * | 2014-02-14 | 2014-07-02 | 华南农业大学 | Molecular marker related to yellow chicken feather and application of molecular marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102094022A (en) * | 2010-08-31 | 2011-06-15 | 李宁 | Gene Hoxc8 related to tassel head characters of chicken and application thereof |
| CN103898102A (en) * | 2014-02-14 | 2014-07-02 | 华南农业大学 | Molecular marker related to yellow chicken feather and application of molecular marker |
Non-Patent Citations (1)
| Title |
|---|
| 李达鉴等.黄羽肉鸡毛囊大小与主翼羽性状遗传参数估计.中国家禽.2021,第43卷(第8期),14-18. * |
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