CN112795667A - Detection method for bovine FRAS1 gene insertion/deletion mutation and application thereof - Google Patents
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Abstract
The invention discloses a detection method of bovine FRAS1 gene insertion/deletion mutation and application thereof. The method comprises the steps of taking cattle whole genome DNA as a template, amplifying partial fragments of cattle FRAS1 gene by PCR, and identifying the genotype of individual FRAS1 gene 15-bp deletion mutation sites by agarose gel electrophoresis. According to the result of the trait association analysis, the genotype of the 15-bp deletion mutation site of the FRAS1 gene is obviously related to the reproductive traits of cows. Therefore, the FRAS1 gene 15-bp deletion mutation site can be used for early marker-assisted selection of cow reproductive traits so as to accelerate the establishment of a cow population with excellent genetic resources.
Description
Technical Field
The invention belongs to the technical field of biotechnology and livestock breeding, and relates to detection and application of bovine FRAS1 gene insertion/deletion (InDel) polymorphism.
Background
Improving the fertility of female livestock is the most direct and effective way to make the cattle breeding industry profitable. However, with the continuous manual selection of high producing cows, the female fertility is gradually reduced in step. Therefore, increasing the reproductive performance of cows is an urgent problem to be solved.
The ovary is the source of cow gonads and germ cells, such as oocytes. Importantly, the development and morphology of the ovaries are closely related to the reproductive performance of cows. The corpus luteum in the ovary is a "temporary gland" whose main task is to synthesize and secrete progesterone, a natural progestational hormone, which has a significant effect on estrogen-stimulated endometrial morphology in the body necessary to maintain pregnancy, or to further atrophy and regress into the white body. Niswender et al showed that, under stimulation by growth hormone and angiogenic factors, luteal cells bind low density lipoprotein cholesterol as a substrate for steroid biosynthesis, eventually forming and secreting progesterone by activating various enzyme systems, and that luteolysis occurs after about 14 days if pregnancy does not occur within one cycle. Therefore, the ovarian related traits and the characteristics of the corpus luteum and the corpus albium can be used as effective indexes for estimating female reproductive capacity, and the selection of key candidate genes for regulating and controlling the related traits is a precondition for improving the reproductive capacity of the cattle by applying molecular marker-assisted selection.
Molecular Marker-assisted Selection (MAS) is widely applied to animal breeding, and can remarkably improve the accuracy and the efficiency of Selection. The molecular marker-assisted selection can rapidly and accurately analyze the genetic composition of individuals from the molecular level, thereby realizing the direct selection of genotypes and the molecular breeding. Insertion/deletion (InDel) mutation is used as an important molecular genetic marker, and has the advantages of wide distribution, high density, large quantity, convenient detection and the like. Compared with other variants, the InDel polymorphism is easier to be directly identified by PCR amplification and agarose gel electrophoresis technology, but as a genetic identification marker, research and application of the InDel marker in breeding of ruminant reproductive traits are few. Cai et al have identified 7 Quantitative Trait Loci (QTL) on 6 chromosomes of cattle based on genome-wide association studies (GWAS). On chromosome 1, 10 candidate genes for two QTLs were identified, while for the remaining chromosomes, different expression of the genes under different studies for each QTL suggested FRAS1, ITGB5, ADCY5 and SEMA5B as candidate genes for the reproductive traits of cows. Other studies have also proposed biologically supported candidate genes, including FRAS 1.
Research shows that FRAS1 encodes ECM protein in embryonic development process and helps to maintain the integrity of embryonic epithelium and mesenchyme, and the mutation of the gene can reduce the reproductive performance of cow obviously. However, reports on the relation between the FRAS1 gene polymorphism and the cattle breeding traits are not found at present.
Disclosure of Invention
The invention aims to provide a detection method of bovine FRAS1 gene insertion/deletion mutation and application thereof, which can rapidly establish a genetic resource population with excellent reproductive traits and accelerate the breeding of the excellent reproductive performance of cattle by simply, rapidly, cheaply and accurately typing insertion/deletion (InDel) polymorphic sites.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting polymorphism of cattle FRAS1 gene InDel comprises the following steps:
taking the genome DNA of a cattle individual to be detected as a template, amplifying a segment containing InDel polymorphic sites of an exon region of a FRAS1 gene by utilizing PCR, carrying out electrophoresis on an amplification product, and identifying the genotype of the InDel polymorphic sites of the individual according to an electrophoresis result; the InDel polymorphic site is selected from a 15-bp deletion mutation site at the position of NC-037333.1 g.340433-340447 of a bovine FRAS1 gene.
Preferably, the amplification primer pair of the PCR is:
an upstream primer: 5'-ACAGAATTCTCTCCAGAGCAATGAA-3'
A downstream primer: 5'-CTGTCTTGGAAGAAACAGTGGC-3' are provided.
Preferably, the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 20s, and 18 cycles, wherein the annealing temperature is reduced by 1 ℃ after each cycle; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 20s, 25 cycles; extending for 10min at 72 ℃; the electrophoresis is performed by using agarose gel with the mass concentration of 2.5%.
Preferably, according to the electrophoresis result, the InDel polymorphic site has three genotypes, wherein the insertion/insertion genotype II shows one stripe of 252bp, the insertion/deletion genotype ID shows two stripes of 252bp and 237bp, and the deletion/deletion genotype DD shows one stripe of 237 bp.
A detection kit for the polymorphism of the cattle FRAS1 gene InDel comprises a primer pair (for example, the upstream primer and the downstream primer) for PCR amplification of 15-bp deletion mutation sites of cattle FRAS1 gene NC-037333.1: g.340433-340447.
The method for detecting the polymorphism of the cattle FRAS1 gene InDel is applied to the cattle molecular marker-assisted selective breeding.
Preferably, the cattle FRAS1 gene NC-037333.1 g.340433-340447 bit 15-bp deletion mutant site is obviously related to the breeding traits of cattle, wherein individuals with the deletion/deletion genotype DD have better properties such as ovary length, corpus luteum number and the like.
The invention has the beneficial effects that:
according to the invention, primers are designed according to InDel polymorphic sites (NC-037333.1: g.340433-340447) in exon regions of the bovine FRAS1 gene, and the genotype of the InDel polymorphic sites can be detected simply, quickly, at low cost and accurately by using bovine genomic DNA as a template through sequence amplification and electrophoretic identification. The result of the relevance analysis of the InDel polymorphic site and the cattle breeding character shows that the DNA marker (InDel marker) for improving the cattle breeding character exists in the InDel polymorphic site detected by the invention, and can be applied to the cattle molecular marker-assisted selective breeding, so that the establishment of a population with excellent breeding character is accelerated, and the improved breeding speed is improved.
Drawings
FIG. 1 is an agarose gel electrophoresis picture of the amplification product of the 15-bp deletion mutation site of bovine FRAS1 gene; wherein M represents Marker.
FIG. 2 is a sequence diagram of PCR amplification products of bovine FRAS1 gene; in this, the boxed part represents the 15-bp deletion sequence CTAAACAAAAACAAC (NC-037333.1: g.340433-340447).
Detailed Description
The invention is described in further detail below with reference to the figures and examples. It should be noted that the present embodiment is only for explaining the present invention, and does not limit the protection scope of the present invention.
The invention utilizes a PCR method to detect the InDel polymorphism which is possibly generated in a cattle population by the g.340433-340447 locus (reference sequence: NC-037333.1) deletion mutation of the cattle FRAS1 gene, and carries out correlation analysis on the genotype and the cattle reproduction traits to verify whether the molecular marker exists and can be used as an auxiliary selection in cattle molecular breeding. The concrete description is as follows.
1. Experimental drugs and reagents
1.1 Biochemical and biological reagents: (ii) Taq DNA polymerase (available from Fermantas, MBI); ② proteinase K (from Huamei bioengineering Co.); ③ Marker I (available from Tiangen Biochemical technology, Beijing, Ltd.).
1.2 general reagents: the common reagent is purchased from Huamei bioengineering company, and is imported subpackaged product, including citric acid, sodium citrate, glucose, Tris, EDTA, NaCl, NaOH, KCl, Na2HPO4、KH2PO4Tris-saturated phenol, chloroform, isoamyl alcohol, absolute ethyl alcohol, sodium acetate, Sodium Dodecyl Sulfate (SDS), Ethidium Bromide (EB), bromophenol blue, dimethyl benzonitrile FF, acetic acid, sucrose, boric acid, agarose, and the like.
1.3 solution and buffer: all solutions and buffers were prepared using deionized ultrapure water. The reagents were prepared according to the molecular cloning protocol of Sambrook et al, autoclaving conditions of 15bf/in (1.034X 10)5Pa)、25min。
1) Solution for extracting tissue-like DNA:
in addition to the common solutions for genomic DNA extraction, the following reagents were prepared:
(ii) 2mol/L NaCl: 11.688g of the extract was dissolved in water, and the volume was adjusted to 100mL, and the solution was autoclaved.
Tissue DNA extract (100 mL): l mol/L Tris-HCl (pH 8.0) L mL, 0.5mol/L EDTA (pH 8.0)20mL, and 2mol/L NaCl 5mL, to a volume of 100 mL.
2) Solutions for agarose gel electrophoresis analysis
(ii) 0.5 × TBE buffer: take 10 XTBE 50mL to 1000 mL. Sample loading buffer solution: contains 0.25% bromophenol blue and 0.25% xylene blue FF, and the solvent is 40.0% (w/v) sucrose aqueous solution.
2. Design of bovine FRAS1 gene mutation site primer
Based on the bovine FRAS1 reference gene sequence (NC _037333.1), a primer pair capable of amplifying a DNA fragment containing the 15-bp deletion mutation site (NC _037333.1: g.340433-340447) of the bovine FRAS1 gene was designed using NCBI, and the sequences of the primer pair were as follows:
an upstream primer: 5'-ACAGAATTCTCTCCAGAGCAATGAA-3' (25nt)
A downstream primer: 5'-CTGTCTTGGAAGAAACAGTGGC-3' (22 nt).
Amplifying a bovine genome by using the primers, wherein an amplification product comprises a 15-bp deletion mutation region of a 73 rd exon of a bovine FRAS1 gene (NC-037333.1), and the insertion/insertion genotype of the deletion mutation region can be amplified to obtain a 252bp strip; the insertion/deletion genotype can be amplified to obtain two banding stripes of 252bp and 237 bp; the deletion/deletion genotype can be amplified to obtain a 237bp band.
PCR amplification of bovine FRAS1 Gene target fragment
3.1 cow ovary sample Collection
The method comprises the steps of collecting 410 parts of ovary samples from individual cows (Holstein cows in China), sampling between 3 months and 2020 months in 2019 (sample collection sample: Xian city in Shaanxi province), and determining morphological phenotypes of the samples, including ovary length and corpus luteum number, in the same group by using unified standards, wherein the ovary length is measured by using a vernier caliper or a double ruler. Considering that the size of the ovaries depends on the stage of the oestrus cycle, only healthy cows of the same age stage (5-6 years) are sampled. According to the comparison result of D-loop regions of mitochondrial DNA, all ovaries can be determined to be from different bovine individuals.
3.2 extraction and isolation of genomic DNA from ovarian sample tissue
Reference is made to the molecular cloning guidelines (2002) compiled by Sambrook et al and to the following: lanxian warrior, genetic variation of important functional genes of goats and the relationship between the genetic variation and economic traits [ D. ] in doctor academic thesis of university of agriculture and forestry in northwest, 2007, Shaanxi Yangling.
3.3 agarose gel electrophoresis detection of DNA
Reference is made to the molecular cloning guidelines (2002) compiled by Sambrook et al.
3.4 purification of DNA
Reference is made to the molecular cloning guidelines (2002) compiled by Sambrook et al.
3.5 spectrophotometric detection of DNA
The OD values of the DNA samples at 260nm and 280nm were measured by an ultraviolet photometer. Calculation of DNA content and OD260/OD280The ratio of (a) to (b). Such as OD260/OD280The ratio is less than 1.6, which indicates that the sample contains more protein or phenol, and purification is required; if the ratio is greater than 1.8, then RNA purification removal should be considered.
DNA concentration (ng/. mu.L) ═ 50 XOD260Value x dilution factor.
After the DNA detection, a certain amount of the DNA was taken out and diluted to 20 ng/. mu.L as a template, and stored at-20 ℃ for later use, and the rest at-80 ℃.
3.6PCR reaction System
The PCR reaction system adopts a mixed sample adding method, namely the total amount of various reaction components is calculated according to the quantity of various components required by each reaction system and the quantity of PCR reaction required by 1 reaction, the reaction components are added into 1 1.5mL centrifuge tube, the centrifuge tubes are mixed fully and evenly and then are subjected to instantaneous centrifugation, the reaction components are subpackaged into 0.2mL Eppendorf PCR tubes, template DNA is added, and PCR amplification is carried out after the instantaneous centrifugation.
And (3) PCR reaction system: 2 xTaq PCR Supermix (including Taq DNA polymerase, dNTPs and reaction buffer, concentration 2 ×) 6.5. mu.L, upstream primer 0.4. mu.L, downstream primer 0.4. mu.L (upstream and downstream primer concentration 10 pmol/. mu.L), genomic DNA 0.5. mu.L (concentration 20 ng/. mu.L), and deionized water 5.2. mu.L, for a total of 13. mu.L.
3.7PCR reaction procedure
1) Pre-denaturation at 95.0 ℃ for 5min, and then entering step 2);
2) denaturation at 94.0 ℃ for 30s, annealing at 68.0 ℃ for 30s (1 ℃ per cycle), extension at 72.0 ℃ for 20s for 18 cycles, and then proceeding to step 3);
3) denaturation at 94.0 ℃ for 30s, annealing at 50.0 ℃ for 30s, and extension at 72.0 ℃ for 20s for 25 cycles;
4) after step 3), extension was carried out at 72.0 ℃ for 10 min.
4. Agarose gel electrophoresis detection
Agarose gel electrophoresis detection is divided into 3 steps: 1) making 2.5% agarose gel, dyeing with nucleic acid dye, spotting 4.0 μ L, and performing 120V electrophoresis for 50-60 min; 2) when the DNA fragments with different molecular weights are clearly separated, imaging in a BIO-RAD Gel Doc 2000 Gel imaging system; 3) and analyzing the InDel polymorphism according to the agarose gel electrophoresis result.
The electrophoresis detection result of the amplified product is shown in FIG. 1, the 7 th lane is Marker (from large to small, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp are sequentially arranged), and the rest lanes are the detection results of the amplified target fragments of different individuals. The result shows that the 15-bp deletion mutation site (NC-037333.1: g.340433-340447) of the 73 rd exon of the bovine FRAS1 gene (NC-037333.1) has three genotypes of II, ID and DD, wherein the II is an insertion/insertion genotype and is represented by a 252bp stripe, the ID is an insertion/deletion genotype and is represented by two stripes of 252bp and 237bp, and the DD is a deletion/deletion genotype and is represented by a 252bp stripe. After the amplified fragment is subjected to sequencing identification, 15-bp deletion mutation is found, and a sequencing peak diagram is shown in FIG. 2.
5. Statistical analysis of genotype and gene frequency of 15-bp deletion mutation site of bovine FRAS1 gene
Genotype frequency refers to the ratio of the number of individuals with a certain genotype for a trait to the total number of individuals in a population:
PYY＝NYY/N
in the formula, PYYRepresents the YY genotype frequency of a certain locus; n is a radical ofYYRepresenting the number of individuals in the population having a YY genotype; and N is the total number of detection groups.
Gene frequency refers to the relative ratio of a certain number of genes in a population to the total number of its alleles:
PY＝(2NYY+NYa1+NYa2+NYa3+NYa4+……+NYan)/2N
in the formula, PYIndicating allele Y frequency, NYYRepresenting the number of individuals in the population having the YY genotype, NYaiRepresenting the number of individuals having the Yai genotype in the population, a 1-an is n mutually different multiple alleles of allele Y.
The genotype frequency, allele frequency and genetic polymorphism index of the 15-bp deletion mutation site of the bovine FRAS1 gene are shown in Table 1 for the collected samples.
TABLE 1 genetic parameter analysis of the 15-bp deletion mutation site of bovine FRAS1 gene
According to the genotype and gene frequency analysis (Table 1), the 15-bp deletion mutation site (NC-037333.1: g.340433-340447) of the bovine FRAS1 gene is an InDel polymorphic site.
6. Correlation analysis of cattle FRAS1 gene InDel site gene polymorphism and ovary-related traits
Genotype data: carrying out agarose gel electrophoresis on the genotype identified after PCR amplification;
the analysis method comprises the following steps: the correlation between the genotype and the breeding trait of the bovine individuals was analyzed by SPSS, and the results are shown in table 2.
TABLE 2 Association analysis of genetic polymorphisms with ovarian-related traits
Note: the same row data mean value is different from the shoulder letter, which indicates that the statistical difference exists
As can be seen from Table 2, the polymorphism of the 15-bp deletion mutation site of the FRAS1 gene has significant influence on the reproductive traits of cows, the 15-bp deletion mutation is significantly related to the ovary length and the corpus luteum number of a cow individual (P <0.05), and the DD genotype of the 15-bp deletion mutation site (NC-037333.1: g.340433-340447) of the FRAS1 gene can be used as a DNA molecular marker of the reproductive traits of cows.
In a word, the invention detects a new InDel polymorphic site (NC-037333.1: g.340433-340447) of the discovered bovine FRAS1 gene by using a PCR amplification method, and performs relevance analysis on the polymorphic site and the reproductive traits of the bovine, finds that the polymorphic site is obviously related to the reproductive traits of the bovine, has a molecular marker serving as auxiliary selection in bovine molecular breeding, can be used for quickly establishing a population with excellent reproductive traits, and further accelerates the speed of fine breed breeding.
<110> northwest agriculture and forestry science and technology university
<120> detection method of bovine FRAS1 gene insertion/deletion mutation and application thereof
<160> 3
<210>1
<211> 25
<212> DNA
<213> upstream primer
<400> 1
acagaattct ctccagagca atgaa 25
<210>2
<211> 22
<212> DNA
<213> downstream primer
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ctgtcttgga agaaacagtg gc 22
<210>3
<211> 15
<212> DNA
<213> NC-037333.1 g.340433-340447 bit deleted sequence
<400> 3
ctaaacaaaa acaac 15
Claims (10)
1. A method for detecting the insertion/deletion polymorphism of the FRAS1 gene of cattle, which is characterized in that: the method comprises the following steps:
taking bovine genome DNA to be detected as a template, amplifying a fragment containing the insertion/deletion polymorphic site of an FRAS1 gene exon region by utilizing PCR (polymerase chain reaction), carrying out electrophoresis on an amplification product, and identifying the genotype of the insertion/deletion polymorphic site according to an electrophoresis result; the insertion/deletion polymorphic sites are selected from 15-bp deletion mutant sites of the bovine FRAS1 gene NC-037333.1 g.340433-340447.
2. The method for detecting the bovine FRAS1 gene insertion/deletion polymorphism according to claim 1, wherein the method comprises the following steps: the PCR amplification primer pair comprises:
an upstream primer: 5'-ACAGAATTCTCTCCAGAGCAATGAA-3'
A downstream primer: 5'-CTGTCTTGGAAGAAACAGTGGC-3' are provided.
3. The method for detecting the bovine FRAS1 gene insertion/deletion polymorphism according to claim 1, wherein the method comprises the following steps: the reaction procedure adopted by the PCR is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 20s, and 18 cycles, wherein the annealing temperature is reduced by 1 ℃ after each cycle; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 20s, 25 cycles; extending for 10min at 72 ℃; the electrophoresis is performed by using agarose gel with the mass concentration of 2.5%.
4. The method for detecting the bovine FRAS1 gene insertion/deletion polymorphism according to claim 1, wherein the method comprises the following steps: according to the electrophoresis result, the insertion/deletion polymorphic site has three genotypes, wherein the insertion/insertion genotype II shows one stripe of 252bp, the insertion/deletion genotype ID shows two stripes of 252bp and 237bp, and the deletion/deletion genotype DD shows one stripe of 237 bp.
5. The application of 15-bp deletion mutation sites of g.340433-340447 of a bovine FRAS1 gene NC-037333.1 in bovine molecular marker-assisted selective breeding.
6. A detection kit for bovine FRAS1 gene insertion/deletion polymorphism is characterized in that: the kit comprises a primer pair for PCR amplification of a 15-bp deletion mutation site of a bovine FRAS1 gene NC-037333.1 g.340433-340447.
7. The kit for detecting the bovine FRAS1 gene insertion/deletion polymorphism according to claim 6, wherein the kit comprises: the primer pair is as follows:
an upstream primer: 5'-ACAGAATTCTCTCCAGAGCAATGAA-3'
A downstream primer: 5'-CTGTCTTGGAAGAAACAGTGGC-3' are provided.
8. The use of the method of detecting an insertion/deletion polymorphism of the bovine FRAS1 gene according to claim 1 in bovine molecular marker-assisted selection breeding.
9. Use according to claim 5 or 8, characterized in that: the 15-bp deletion mutant site of the bovine FRAS1 gene NC-037333.1 g.340433-340447 is obviously related to the reproductive traits of cattle, wherein individuals with deletion/deletion genotype DD have better reproductive traits.
10. Use according to claim 9, characterized in that: the reproductive traits are selected from one or two of ovary length and corpus luteum number.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703293A (en) * | 2022-03-22 | 2022-07-05 | 西北农林科技大学 | Application of InDel marker of cattle CRY1 gene in early selection of reproductive traits |
| CN116200472A (en) * | 2023-04-04 | 2023-06-02 | 西北农林科技大学 | Application of cattle SCN9A gene InDel marker in early selection of meat quality traits |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103725773A (en) * | 2012-10-10 | 2014-04-16 | 杭州普望生物技术有限公司 | Technology for identifying HBV (hepatitis B virus) gene integration sites and recurrently targeted genes in host genome |
| CN109182539A (en) * | 2018-10-09 | 2019-01-11 | 西北农林科技大学 | A kind of detection method and its application of ox IGF1R gene insertion/deletion |
-
2021
- 2021-03-22 CN CN202110302857.9A patent/CN112795667B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103725773A (en) * | 2012-10-10 | 2014-04-16 | 杭州普望生物技术有限公司 | Technology for identifying HBV (hepatitis B virus) gene integration sites and recurrently targeted genes in host genome |
| CN109182539A (en) * | 2018-10-09 | 2019-01-11 | 西北农林科技大学 | A kind of detection method and its application of ox IGF1R gene insertion/deletion |
Non-Patent Citations (3)
| Title |
|---|
| 1000_BULL_GENOMES_1000_BULL_GENOMES_RUN2: "Variant Summary for rs453892138" * |
| A. SLAVOTINEK 等: "Mutation Analysis of the FRAS1 Gene Demonstrates New Mutations in a Propositus With Fraser Syndrome" * |
| JOLANTA E. PITERA 等: "Generation of Mice With a Conditional Null Fraser syndrome 1 (Fras1) Allele" * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703293A (en) * | 2022-03-22 | 2022-07-05 | 西北农林科技大学 | Application of InDel marker of cattle CRY1 gene in early selection of reproductive traits |
| CN114703293B (en) * | 2022-03-22 | 2023-06-02 | 西北农林科技大学 | Application of InDel marker of cattle CRY1 gene in early selection of reproductive traits |
| CN116200472A (en) * | 2023-04-04 | 2023-06-02 | 西北农林科技大学 | Application of cattle SCN9A gene InDel marker in early selection of meat quality traits |
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