CN112852979B - Molecular marker related to duck breast muscle fiber diameter character and application thereof - Google Patents
Molecular marker related to duck breast muscle fiber diameter character and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker related to duck breast muscle fiber diameter character and application thereof. The invention provides a method for identifying or assisting in identifying diameter characters of breast muscle fibers of ducks, which comprises the following steps: detecting the genotype of the duck to be detected based on the specific SNP, wherein the diameter of the pectoralis muscle fiber of the CC genotype duck is larger than that of the CT genotype duck, and the diameter of the pectoralis muscle fiber of the CT genotype duck is larger than that of the TT genotype duck. The specific SNP is 11396601 th nucleotide of No. 3 chromosome of duck genome. The SNP molecular marker closely related to duck breast muscle fiber diameter characters is obtained through whole genome association analysis, and the early selection of the duck breast muscle fiber diameter characters is carried out by utilizing the SNP molecular marker, so that the production cost can be saved, the breast muscle yield is increased, the genetic progress is accelerated, the breeding of ducks is better served, and the method has great economic application value and scientific research value.
Description
Technical Field
The invention belongs to the technical field of animal genetic breeding, relates to a molecular marker assisted selection technology, and particularly relates to a molecular marker related to duck breast muscle fiber diameter characters and application thereof.
Background
With the improvement of living standard, the demand of people on meat products is not limited to quantity, and the demand on meat quality is higher and higher. The most important component of animal body is skeletal muscle, which accounts for 40% of animal body, while muscle fiber is the main component of skeletal muscle, and the meat quality and the histological characteristics of muscle fiber are closely related.
The number of skeletal muscle fibers in animals is essentially established during embryonic development, and the increase in muscle mass is due to hypertrophy of skeletal muscle fibers after birth. This means that thickening the muscle fiber diameter directly affects muscle yield. In addition, the diameter of muscle fiber has a high positive correlation with the tenderness of meat, and the increase of the diameter of muscle fiber can promote the tenderness of meat, which is one of important indexes for evaluating meat quality and plays an important role in meat quality.
The selection of the traditional breeding method for increasing the diameter of the breast muscle fiber of the Beijing duck has already made a certain progress, but for local varieties in China, the improvement space of the diameter of the breast muscle fiber is still large. The molecular marker assisted breeding technology is a new breeding technology formed in recent years and has been widely applied to breeding new varieties of animals and plants. However, at present, no molecular marker related to the increase of the diameter of the muscle fiber of the breast muscle of the duck exists, which greatly hinders the development of the meat duck industry.
Disclosure of Invention
The invention aims to provide a molecular marker related to duck breast muscle fiber diameter characters and application thereof.
The present invention provides an application of a substance for detecting a specific SNP, which is (d1) or (d 2):
(d1) identifying or assisting in identifying the diameter character of the breast muscle fiber of the duck;
(d2) preparing a kit for identifying or assisting in identifying the diameter character of the breast muscle fiber of the duck.
The invention also provides a method for identifying or assisting in identifying the diameter character of the breast muscle fiber of the duck, which comprises the following steps: detecting the genotype of the duck to be detected based on the specific SNP, wherein the diameter of the pectoralis muscle fiber of the CC genotype duck is larger than that of the CT genotype duck, and the diameter of the pectoralis muscle fiber of the CT genotype duck is larger than that of the TT genotype duck.
The invention also provides a duck breeding method, which comprises the following steps: detecting the genotype of the duck to be detected based on the specific SNP; and (3) selecting the CC genotype duck for breeding.
The invention also protects specific DNA molecules.
The invention also protects the application of the specific DNA molecule in identification or auxiliary identification of the diameter property of the breast muscle fiber of the duck.
The invention also protects a specific primer pair, which consists of a primer F and a primer R;
primer F is (b1) or (b2) as follows:
(b1) a single-stranded DNA molecule shown as SEQ ID NO. 2;
(b2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in (b1) and have the same functions;
the primer R is (c1) or (c2) as follows:
(c1) a single-stranded DNA molecule represented by SEQ ID NO. 3;
(c2) and (c1) is replaced and/or deleted and/or added by one or more nucleotides, and the DNA molecule has the same function.
The invention also protects a kit comprising the specific primer pair. The kit may also include conventional reagents for PCR amplification. The kit may also include sequencing conventional reagents.
The invention also protects the application of the specific primer pair or the kit, which is (f1) or (f 2):
(f1) identifying or assisting in identifying the diameter character of the breast muscle fiber of the duck;
(f2) and (5) breeding ducks.
Any of the specific SNPs are as follows (g1) or (g 2):
(g1) 332 th nucleotide of a specific DNA molecule in duck genome DNA;
(g2) 11396601 th nucleotide of No. 3 chromosome of duck genome.
The specific SNP is C/T polymorphism.
Any one of the above specific DNA molecules is (a1) or (a2) or (a3) or (a 4):
(a1) 1, DNA molecule shown in SEQ ID NO;
(a2) a DNA molecule derived from duck and having 10 or less different nucleotides from (a 1); the 10 or fewer different nucleotides do not include nucleotide 332;
(a3) DNA molecules which are derived from ducks and have more than 98 percent of identity with the DNA molecules shown in SEQ ID NO. 1;
(a4) the target sequence of the specific primer pair in the duck genome.
The substance for detecting a specific SNP may specifically be the specific primer pair.
The method for detecting the specific SNP-based genotype of the duck to be detected specifically comprises the following steps: taking the genomic DNA of the duck to be detected as a template, carrying out PCR amplification by adopting a specific primer pair, and sequencing the PCR amplification product.
The invention also provides an SNP molecular marker related to duck breast muscle fiber diameter character, which is characterized in that: the SNP molecular marker is located at 11396601 th nucleotide of No. 3 chromosome of duck genome and is C/T polymorphism.
The invention also protects the application of the SNP molecular marker in duck breeding.
The breeding aim is to breed individuals with large diameter of pectoralis muscle fibers (thick diameter of pectoralis muscle fibers).
The breeding aim is to breed a group with large diameter of pectoralis muscle fiber (thick diameter of pectoralis muscle fiber).
The breeding aim is to breed a variety with large diameter of pectoralis muscle fiber (thick diameter of pectoralis muscle fiber). .
In the breeding, individuals with CT genotypes and TT genotypes are eliminated.
In the breeding, individuals of CC genotypes are retained.
In the application, TT genotype breeding duck individuals are eliminated, and CT genotype and CC genotype individuals are reserved to improve the frequency of allele C in duck groups, so that the diameter of duck pectoralis muscle fiber is increased.
In SEQ ID NO. 1, the specific SNP is represented by Y, which represents C or T.
Any one of the ducks is a meat duck.
The meat duck is a small-body meat duck.
The meat duck can be Beijing duck, Shaoxing duck, Liancheng white duck or Putian black duck.
The SNP molecular marker closely related to duck breast muscle fiber diameter characters is obtained through whole genome association analysis, and the early selection of the duck breast muscle fiber diameter characters is carried out by utilizing the SNP molecular marker, so that the production cost can be saved, the breast muscle yield is increased, the genetic progress is accelerated, the breeding of ducks is better served, and the method has great economic application value and scientific research value.
Drawings
FIG. 1 is a graph showing the results of the association analysis of all genes in example 1.
FIG. 2 is a partial sequencing result of an exemplary sample of test animals of each genotype.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.
The composition of the raw materials of the feed used in the examples is shown in table 1. The nutrition level in the feed is as follows (% represents the mass percentage): 17.59% of crude protein, 12.64MJ of metabolizable energy, 0.91% of lysine, 0.4% of methionine, 0.99% of calcium and 0.42% of phosphorus.
TABLE 1 feed raw Material composition
The premix is provided for each kilogram of feed: 8000IU of vitamin A, 33000 IU of vitamin D, 20IU of vitamin E, vitamin K32 mg of vitamin B, 10.65mg of vitamin B, 22.21mg of vitamin B, 3.51mg of pantothenic acid, 19.8mg of nicotinic acid, 3.25mg of pyridoxine, 0.20mg of biotin, 0.28mg of folic acid, 120.02mg of vitamin B, 80mg of manganese, 60mg of iron, 10mg of copper, 60mg of zinc, 1.0mg of iodine and 0.3mg of selenium.
Example 1 determination of the correlation of specific SNPs with the diameter of the fibers of the pectoral muscle
Obtaining a test population
Crossing the green-head wild duck (male duck) and the Beijing duck (female duck) to obtain an orthogonal F1 generation group. The green wild duck (female duck) and the Beijing duck (male duck) are hybridized to obtain the population of the reverse crossing F1 generation. The orthogonal F1 generation population was freely mated with the reverse crossing F1 generation population to give 300F 2 individuals.
Second, measurement of diameter of fiber of pectoral muscle
The experimental groups were fed with the same feed and under the same conditions, and the whole process was free to eat and drink water.
The diameter of pectoral muscle fibers at the age of 56 days was measured for each individual. The detection method comprises the following steps: the duck breast muscle is taken from the unified part and put into 4% formaldehyde solution for fixation, then paraffin sections are made, a digital microscope is adopted for image acquisition of breast muscle fiber morphology, and IPP software is used for acquiring breast muscle fiber histological morphology indexes to obtain muscle fiber diameter.
Thirdly, obtaining SNP molecular marker
1. Blood sample collection
Collecting the wing vein blood of each individual by heparin sodium anticoagulation blood collection tube, and preserving at-20 ℃.
2. Whole blood genomic DNA extraction
The genomic DNA was extracted from each blood sample.
3. Genotyping
Genome DNA is taken, a Hiseq X-Ten sequencing platform of Illumina company is adopted to carry out whole genome individual re-sequencing, the sequencing depth of each individual is about 5X, and the specific method refers to a standard operation flow provided by Illumina company. After the off-line data is subjected to quality control, two bioinformatics software of BWA and GATK are adopted for distribution to carry out sequence comparison and genotype extraction.
Genome-wide association analysis of diameter of pectoralis muscle fibers
Genome-wide correlation analysis of pectoralis muscle fiber diameter and genotype statistical analysis was performed using a compressed mixed linear model of the EMMAX software. The results of the correlation analysis are shown in FIG. 1, with the abscissa representing the chromosome number and the ordinate representing-log10(P)。
An SNP which is significantly related to the diameter trait of the fibers of the pectoralis muscles is found and is positioned at 11396601 th nucleotide of No. 3 chromosome, so that the SNP is named as 'chr 3:11396601 site SNP', and is subsequently called as a specific SNP for conciseness. The specific SNP is C/T polymorphism.
A pair of primers was designed based on the specific SNP, consisting of F1 and R1. The target sequences of F1 and R1 in the duck genome DNA are 708bp, and the specific SNP is located at the 332 th nucleotide of the target sequence.
F1(SEQ ID NO:2)：5'-CAACCTTAACTGTGGCACCT-3'；
R1(SEQ ID NO:3)：5'-TTCAAAGCCTCCTCCATTAG-3'。
Example 2 application of specific SNP in genetic improvement of diameter trait of pectoralis muscle fiber
Test animals: 160 Beijing ducks.
Detection based on specific SNP locus genotype
1. Blood sample collection
Collecting the wing vein blood of each test animal by using a heparin sodium anticoagulant blood collection tube, and preserving at-20 ℃.
2. Whole blood genomic DNA extraction
The genomic DNA was extracted from each blood sample.
3. Detection of genotype
And (3) performing PCR amplification by using the genomic DNA as a template and adopting a primer pair consisting of F1 and R1, and then sequencing the PCR amplification product.
The results show that 160 experimental animals all obtained 708bp PCR amplification products.
Based on the specific SNPs, 160 test animals were classified into three genotypes, TT genotype (43 test animals), CC genotype (51 test animals) and CT genotype (66 test animals).
The sequencing result of one of the test animals is shown in SEQ ID NO. 1 (based on the CC genotype of the specific SNP). The sequencing results of other test animals showed 98% or more similarity to SEQ ID NO. 1 (the difference in the PCR amplification product was 10 or less nucleotides except for the specific SNP).
Partial sequencing results for an exemplary sample of test animals of each genotype are shown in FIG. 2.
Measurement of diameter of fiber of breast muscle of duck
All test animals were fed with the same feed and under the same conditions, and were fed with free access to food and water throughout the entire process.
The diameter of the pectoral muscle fibers at the age of 56 days was measured for each test animal. The detection method comprises the following steps: the duck breast muscle is taken from the unified part and put into 4% formaldehyde solution for fixation, then paraffin sections are made, a digital microscope is adopted for image acquisition of breast muscle fiber morphology, and IPP software is used for acquiring breast muscle fiber histological morphology indexes to obtain muscle fiber diameter.
The diameter of the fibers of the muscles of the pectoralis of the test animals of each genotype (in μm) was counted, and the results are shown in Table 2.
TABLE 2
Correlation analysis of genotype and diameter of pectoralis muscle fiber
Statistical analysis is carried out by adopting the GLM process of the SAS statistical analysis software package, variance statistical analysis is carried out on the genotype of the duck group to be tested and the diameter character of the pectoralis muscle fiber according to the generalized linear model, and the difference is obvious when the P-value is less than 0.05.
A statistical analysis model: y ═ μ + G + e;
wherein: y represents an individual phenotype value; μ represents the population mean; g represents a genotype effect; e represents the residual effect.
The results show that the diameter difference of the pectoral muscle fibers of the three genotypes of ducks is very obvious (P-value is 6.76 multiplied by 10)-6). The diameter of the muscle fiber of the breast muscle of the CC genotype duck is larger than that of the muscle fiber of the CT genotype duck and that of the TT genotype duck, and the diameter of the muscle fiber of the CT genotype duck is larger than that of the TT genotype duck.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> molecular marker related to duck breast muscle fiber diameter character and application thereof
<130> GNCYX211154
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> Anatinae
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caaccttaac tgtggcacct aacaacagaa tgcaagagcc aagagccatt ccatgcaaga 60
atgatggatt gagtgaaagc atgctaaata agataggcat tgccaatggc ctttatttag 120
cataaagata agtagaagct aaaccctttt ttgttttgga gtggtataaa accacagttt 180
ttgcctttaa gagctcacat gtgcatttgt gcctatgcac acacttgtat gtgcacacac 240
agaacagcct ccccttttaa gcacccagcc actccaaaag ccaaaggcac actctgaagg 300
tcacttgaaa ctctttaata aatatttttt gytggtatat gaatataaac acaggaagat 360
gggtattact tggcaggaaa atattcgtga acaaaatccg accacaggag aaacaattgt 420
ctgccttttg gctgtacttt aagaaaacat ttcctatttt ttcctgaacg caatatgaat 480
taaagacatt gtgcatattt ttgtcattca aaataatatc aagtgtgact tcaatctctc 540
ccagctggga catccatgat gattgtggat attggtctac taactgtgcc aagaagaggt 600
aggttgtgtt tttcctggga gataagttgg aaagtaataa ttcttttttg gagatacaga 660
gccttagata taaaatctac agtaacagct aatggaggag gctttgaa 708
<210> 2
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<213> Artificial Sequence (Artificial Sequence)
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caaccttaac tgtggcacct 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttcaaagcct cctccattag 20
Claims (5)
1. The application of the substance for detecting specific SNP is (d1) or (d 2):
(d1) identifying or assisting in identifying the diameter character of the breast muscle fiber of the duck;
(d2) preparing a kit for identifying or assisting in identifying the diameter character of the breast muscle fiber of the duck;
the specific SNP is 332 th nucleotide of a specific DNA molecule in duck genome DNA; the specific DNA molecule is shown as SEQ ID NO. 1;
the duck is Beijing duck.
2. A method for identifying or assisting in identifying diameter characters of breast muscle fibers of ducks comprises the following steps:
detecting the genotype of the duck to be detected based on the specific SNP, wherein the diameter of the pectoralis muscle fiber of the CC genotype duck is larger than that of the CT genotype duck, and the diameter of the pectoralis muscle fiber of the CT genotype duck is larger than that of the TT genotype duck;
the specific SNP is 332 th nucleotide of a specific DNA molecule in duck genome DNA; the specific DNA molecule is shown as SEQ ID NO. 1;
the duck is Beijing duck.
3. A method for breeding ducks comprises the following steps:
detecting the genotype of the duck to be detected based on the specific SNP; selecting CC genotype ducks for breeding;
the specific SNP is 332 th nucleotide of a specific DNA molecule in duck genome DNA; the specific DNA molecule is shown as SEQ ID NO. 1;
the duck is Beijing duck.
4. The specific DNA molecule is shown as SEQ ID NO. 1.
5. The use of the specific DNA molecule of claim 4 in the identification or auxiliary identification of the diameter character of the breast muscle fiber of the duck; the duck is Beijing duck.
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| CN108165639B (en) * | 2018-03-12 | 2019-01-01 | 中国农业科学院北京畜牧兽医研究所 | One kind molecular labeling relevant to fleshy duck fodder transformation efficiency character and its application |
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