CN110468212B - SNP (single nucleotide polymorphism) site related to content of fatty acid C14:0 on chromosome 19 of meat Simmental cattle and application - Google Patents
SNP (single nucleotide polymorphism) site related to content of fatty acid C14:0 on chromosome 19 of meat Simmental cattle and application Download PDFInfo
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Abstract
The invention provides a method for preparing a new type of beef Simmental cattle chromosome 19 with fatty acid C14:0 content related SNP locus and application, wherein the SNP marker locus is 51333374 th nucleotide locus on chromosome 19 of UMD3.1 version of the international bovine reference genome, and the base of the locus is T or A. By optimizing the dominant allele of the SNP, the invention can increase the frequency of the dominant allele generation by generation, and improve the fatty acid C14:0 content, thereby accelerating the genetic improvement progress of the beef cattle and effectively improving the economic benefit of beef cattle breeding.
Description
Technical Field
The invention relates to an SNP locus related to fatty acid shape on a chromosome 19 of a meat Simmental cattle and application thereof.
Background
China has been breeding for nearly 20 years for meat Simmental cattle. At present, the meat Simmental cattle accounts for 60 to 70 percent of various hybridization improved cattle groups in China and accounts for more than 50 percent of the stock ratio of Chinese beef cattle. And the descendant determination system of beef cattle in China is just established and still needs to be gradually improved, so that in order to accelerate the molecular breeding process of beef cattle in China, the significant sites influencing the fatty acid character of the western siemens-tals cattle in China are identified by adopting whole genome association analysis. The content of fatty acid in muscle is an important index for evaluating meat quality, the components of the fatty acid are significant for improving the meat quality flavor, and the meat quality flavor is one of the most important main factors for evaluating the meat quality of consumers. Research results show that fatty acid components often belong to low-medium heritability, and C14: the heritability for the 0 content is relatively high, about 0.54. Therefore, the genetic means is utilized to apply the siemens tauro fatty acid C14: the 0 content is preferably modified. Fatty acid C14: the 0 content is a quantitative trait controlled by a plurality of genes, and a great deal of influence fatty acid C14 exists on the bovine genome: quantitative Trait Loci (QTLs) of 0 content. Currently, methods utilizing genome-wide association analysis (GWAS) have identified more than many genes related to fatty acid C14 on the bovine genome: 0-content-related QTLs and a large number of Single Nucleotide Polymorphisms (SNPs), wherein the addition of SNPs having a significant effect to molecular Marker Assisted Selection (MAS) and Genomic Selection (GS) can significantly increase the fatty acid C14:0, and further increasing the fatty acid C14:0 content, improves the meat quality and flavor of the beef and enhances the market competitiveness of commercial beef production of breeding enterprises.
The whole genome association analysis is an analysis method for identifying the relationship between the influence phenotype and the genotype by a statistical analysis strategy based on linkage disequilibrium among SNPs, and plays an important role in identifying molecular markers influencing important economic traits of cattle. The invention identifies that the influence on meat Simmental bovine fatty acid C14 by GWAS analysis strategy: 0, for use in molecular marker assisted selection and in genome selection, selection for increasing the expression of fatty acid C14: the 0 content favorable genotype is reserved, so that the gene frequency of the dominant allele is improved generation by generation, the breeding improvement process of the cattle can be accelerated, and great economic benefit is brought to the breeding of the beef cattle.
Disclosure of Invention
In order to achieve the above object, the primary object of the present invention is to provide a method for synthesizing a protein on chromosome 19 of a bovine animal with fatty acid C14:0, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1, wherein M in the sequence is T or a, resulting in a fatty acid of bovine C14: the content of 0 is different.
The molecular marker is positioned on a nucleotide sequence on a chromosome 19 of a meat Simmental cattle, and the SNP locus of the molecular marker is SEQ ID NO: 1 nucleotide mutation of T119-a119 at the position marked with the sequence 119; the SNP locus of the molecular marker corresponds to the 51333374 th T & gtA mutation on the chromosome 19 of the reference sequence of the international bovine genome UMD3.1 version.
The invention also aims to provide the molecular marker for screening high fatty acid C14:0, specifically, detecting the molecular marker of claim 1 on the chromosome 19 of the cattle, wherein the 119 th single nucleotide at the 5' end of the molecular marker is T or A, and eliminating A retains T. The cattle is selected from western siemens cattle resource groups for meat in pasture of inner Mongolia Silo Allium management area.
It is another object of the present invention to provide a method for identifying the above-mentioned affecting bovine fatty acid C14:0 content of a molecularly labeled primer pair, the nucleic acid sequence of the primer pair is as follows:
the sequence of the forward primer is shown as SEQ ID NO: 2 is shown in the specification;
the reverse primer sequence is shown as SEQ ID NO: 3, respectively.
The primer pair is used for identifying the primer pair affecting the expression of the bovine fatty acid C14:0 content.
The application of the primer pair in the selection of bovine genome is provided.
The primer pair is used for increasing the ratio of the fatty acid C14:0 content.
The invention aims to provide a method for genetic improvement of cattle, which comprises the following steps: determining the above-described impact of cattle in a cattle resource population on bovine fatty acid C14:0, and making corresponding selections according to the molecular markers: selecting cattle individuals with TT, TA and AA genotypes at 51333374 th site on chromosome 19 of UMD3.1 version of international cattle reference genome, eliminating cattle individuals with AA genotypes at 51333374 th site, and increasing the frequency of allele T at the site by generations, so as to increase the fatty acid C14 of the offspring cattle: 0 content.
Compared with the prior art, the invention has the following advantages and effects:
the invention researches and determines that the fatty acid C14:0, and verifying that the molecular marker has a molecular marker related to the content of the fatty acid C14:0, finally establishing an efficient and accurate genome selective breeding technology, and applying the efficient and accurate genome selective breeding technology to improve the fatty acid C14:0, thereby increasing the fatty acid C14:0, improving the meat quality, and further increasing the market competitiveness of breeding enterprises.
Drawings
Fig. 1 shows that the meat simmental cattle has a fatty acid C14 on chromosome 19: genome-wide association analysis (GWAS) manhattan plot of 0 content; wherein: the abscissa represents the chromosome number of cattle; the ordinate represents the-logP value.
FIG. 2 shows fatty acid C14 of different genotypes of Simmental cattle: 0 content.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The above object of the present invention is specifically achieved by:
example 1
1. Laboratory animal
The experimental cattle groups used in the invention are all from Ximantal cattle 663 in pasture of Wula Gai management area of Guo of Heileo of inner Mongolia, and are meat Simmental cattle resource groups established by cattle genetic breeding innovation team of Beijing animal veterinary institute of Chinese academy of agricultural sciences.
663 head Simmental cattle for meat in the resource group are selected in the experiment. The Simmental cattle resource population for the meat is expanded every year, and newly added individuals generally go through 3 stages of birth, fattening and slaughtering. After the calf born in 3-5 months per year is stocked and managed for a period of time, the calf genetic breeding innovation team performs unified birth weight and body size measurement in the same year in 7 months, and simultaneously performs measurement on the basic cow. And uniformly and intensively fattening young cattle of 5-9 months of age in the same year in 10 months, collecting phenotype data of growth and development traits, and simultaneously carrying out genotyping on Illumina Bovine HD chips to obtain genotype data. When the fattening period of all individuals reaches 10-12 months, namely about 11 months in the next year, all meat is slaughtered in batches by Simmental cattle. The slaughtering process is strictly executed according to meat procurement specifications, and slaughtering data, carcass data and meat quality data are strictly measured according to the requirements of GB/T27643 plus 2011 guidelines for measuring carcass traits and meat quality traits after slaughter.
2. Sample collection
Collecting venous blood 50ml of all individuals of the cattle group by using a blood collection tube, and storing the venous blood in a refrigerator at the temperature of 80 ℃ below zero for later use. 3. SNP (single nucleotide polymorphism) judgment of cattle whole genome 770K high-density chip
Collecting 50ml venous blood from each individual of 663 meat Simmental cattle selected from the above resource groups, extracting whole genome DNA by standard phenol-chloroform method, and accurately determining DNA concentration and OD ratio (OD260/280, OD260/230) of each sample by Nanodrop2000/2000C nucleic acid protein detector. And detecting qualified DNA samples by a NanoDrop2000/2000C nucleic acid protein detector, and diluting the DNA to about 50 ng/. mu.L according to the detected concentration. And mixing 6 mu l of the extracted DNA sample to be detected with 2 mu l of Loading Buffer, Loading the mixture into 1% agarose gel, carrying out electrophoresis for 25min under the voltage of 150V, observing and photographing under an ultraviolet spectrophotometer and gel imaging equipment, and observing the integrity of the DNA.
DNA samples were sent to Neuggium Biotechnology (Shanghai) Co., Ltd and genotype determination of cattle Whole genome Illumina Bovine HD chip 770K SNP chip (Illumina, USA) was carried out according to the company standard procedures. Quality control is carried out on all 770K chip scanning typing data of the sample by utilizing PLINK v1.90 software, the rejection rate is lower than 90%, the family Mendelian error rate is higher than 0.1, the minimum allele frequency is lower than 0.05, and the Hardy-Weinberg equilibrium significance level is higher than 10-6Finally, 671,204 effective genotype data of the SNPs are obtained.
4. Genome-wide association (GWAS) analysis
In order to eliminate the population stratification effect, the GWAS analysis is carried out by adopting single-point regression analysis of a linear mixed model and combining with an R language GenABEL software package, and the stratification effect is corrected by utilizing the similarity of genomes among individuals in an analysis model. Determination of SNP to fatty acid C14 using Bonferrini method: 0 content association degree significance threshold, wherein the genome level significance threshold is 0.05 divided by the number of effective SNP loci, namely the genome significance level threshold is 7.45e-8, namely 0.05/671,204 (effective SNP number); the chromosome level significance threshold was 1 divided by the number of effective SNP sites, i.e., the chromosome significance threshold was 1.49e-6, i.e., 1/671,204 (effective SNP number).
The GWAS analysis results are shown in fig. 1. As can be seen from fig. 1, the presence of fatty acid C14 in chromosome 19 of the meat simmental cattle significantly affected: 0 sites, the most strongly associated SNP was g.119t > a (P ═ 1.80E-10).
5. Different genotypes and fatty acid C14: correlation analysis of 0-content phenotype
According to Table 1, the SNP site g.119T > A and fatty acid C14 of the molecular marker are shown as follows: the 0 content is very significantly related (P < 0.001), which indicates that the molecular marker significantly affects the fatty acid C14 of cattle: 0 content, which can be increased by the aid of selection for this SNP site in cattle, thereby increasing the fatty acid C14:0 content, thereby accelerating the breeding process of the target character.
Further, as shown in Table 1, fatty acid C14 of TT type and TA type, compared with AA type: the content of 0 is high, which indicates that the AA type cattle individual has no toxicity to the screened fatty acid C14: the content of 0 is unfavorable, so TT and TA type cattle are preferentially reserved. Fatty acid C14: the content of 0 is an important index for measuring the meat quality of the cattle, and the content of the fatty acid C14 of the cattle is increased: the content of 0 is beneficial to improving the meat quality and flavor of the beef, thereby increasing the market competitiveness. Therefore, in the breeding process, AA type cattle need to be gradually eliminated, TT and TA type cattle are preferentially reserved, and the frequency of the allele T at the site is increased generation by generation.
Table 1 molecular markers SNP sites g.119t > a and fatty acid C14: correlation of 0 content
6. Amplification and sequencing of DNA sequences of interest
(1) Primer design
Download the sequence of SEQ ID NO on chromosome 19 of cattle via Ensembl website (http:// asia. ensemble. org/index. html): 1. And primers were designed using primer premier 6.0, primer design software.
The DNA sequences of the designed primers are shown below:
p001 forward direction: 5'-CCTGCACAATGAGGGACCAG-3' the flow of the air in the air conditioner,
p002 reverses: 5'-TGTCCGACTCTTTGCAACCC-3', respectively;
(2) PCR amplification
To a 10uL reaction system, 1uL DNA template, 3.4uL double distilled water, 2 Xtag PCR StanMix with loading Dye 5uL, and 0.3uL each of primers P001 and P002 were added. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 45s, 35 cycles, and final extension at 72 ℃ for 5 min.
(3) DNA sequencing
DNA sequencing identification: the two reactions of the gene fragment were measured in Beijing Biotechnology technology Ltd. The measured sequence was compared with the NCBI genomic sequence to obtain the mutation of the corresponding SNP site. The sequencing results are shown below:
note: m marked in the sequence listing is a mutation site and is shown by underlining (the mutation base is shown in parentheses, and is an allelic mutation), and the head and the tail of the sequence are shown in bold as the designed primer sequence position.
7. Molecular marker SNP site g.119T & gtA effect analysis
By auxiliary selection of molecular markers, cattle with AA as a genotype in a colony are eliminated, and the fatty acid C14:0 content, thereby improving the meat quality and bringing more economic benefits for enterprises.
The invention relates to a method for preparing a polypeptide shown in SEQ ID NO: 1, detecting 119 th base mutation site in the sequence, and primarily performing the detection of the genotype of the mutant and the mutation site of bovine fatty acid C14: the application of the correlation analysis between 0 content provides a new molecular marker for the bovine molecular marker-assisted selection and the genome selection.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (4)
1. The SNP marker on the chromosome 19 of the meat Simmental cattle is characterized in that the SNP marker increases the expression level of the fatty acid C14:0, wherein the sequence of the SNP marker is as shown in SEQ ID NO: 1, the sequence shown in SEQ ID NO: 1 is T or A from the 119 th base of the 5' end, forming 3 genotypes of cattle with TT type, TA type and AA type, wherein the fatty acid C14 of the TT type and the TA type is more than that of the AA type: 0 content is high, so AA type cattle are gradually eliminated in the breeding process, TT and TA type cattle are reserved, the frequency of allele T of the locus is improved generation by generation, and the fatty acid C14 of the offspring cattle is improved: 0 content; the cattle are western Mengtal cattle for meat.
2. A method for increasing the fatty acid content of meat Simmental beef C14: 0-content process, characterized in that it comprises the following steps: (1) extracting the genomic DNA of the cattle to be detected; (2) and carrying out PCR amplification on the genomic DNA of the cattle to be detected by adopting a primer pair so as to obtain a PCR amplification product, wherein the nucleic acid sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; (3) sequencing the PCR amplification product so as to obtain a sequencing result; (4) and determining the genotype of the SNP marker of the cattle to be detected based on the sequencing result, wherein the sequence of the SNP marker is shown as SEQ ID NO: 1, the sequence shown in SEQ ID NO: 1 is T or A from the 119 th base of the 5' end, forming 3 genotypes of cattle with TT type, TA type and AA type, wherein the fatty acid C14 of the TT type and the TA type is more than that of the AA type: 0 content is high, so AA type cattle are gradually eliminated in the breeding process, TT and TA type cattle are reserved, the frequency of allele T of the locus is improved generation by generation, and the fatty acid C14 of the offspring cattle is improved: 0 content; the cattle to be detected are western Mengtal cattle for meat.
3. A primer pair for detecting SNP markers on chromosome 19 of meat Simmental cattle is disclosed, wherein the primer pair is used for increasing the ratio of fatty acid C14:0, wherein the nucleic acid sequence of the primer pair is as shown in SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the sequence of the SNP marker is shown as SEQ ID NO: 1, the sequence shown in SEQ ID NO: 1 is T or A from the 119 th base of the 5' end, forming 3 genotypes of cattle with TT type, TA type and AA type, wherein the fatty acid C14 of the TT type and the TA type is more than that of the AA type: 0 content is high, so AA type cattle are gradually eliminated in the breeding process, TT and TA type cattle are reserved, the frequency of allele T of the locus is improved generation by generation, and the fatty acid C14 of the offspring cattle is improved: 0 content; the cattle are western Mengtal cattle for meat.
4. A kit for detecting SNP markers on chromosome 19 of meat Simmental cattle is disclosed, wherein the SNP markers on the chromosome are expressed in the following steps of increasing the fatty acid C14:0, characterized in that the kit comprises a primer pair, and the nucleic acid sequence of the primer pair is shown in SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the sequence of the SNP marker is shown as SEQ ID NO: 1, the sequence shown in SEQ ID NO: 1 is T or A from the 119 th base of the 5' end, forming 3 genotypes of cattle with TT type, TA type and AA type, wherein the fatty acid C14 of the TT type and the TA type is more than that of the AA type: 0 content is high, so AA type cattle are gradually eliminated in the breeding process, TT and TA type cattle are reserved, the frequency of allele T of the locus is improved generation by generation, and the fatty acid C14 of the offspring cattle is improved: 0 content; the cattle are western Mengtal cattle for meat.
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| CN112195253B (en) * | 2020-10-28 | 2022-07-05 | 中国农业科学院北京畜牧兽医研究所 | SNP (Single nucleotide polymorphism) locus for increasing content of fatty acid C14:0 in chicken and method for breeding high-quality chicken strain by using SNP locus |
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| CN101405404A (en) * | 2002-12-31 | 2009-04-08 | 迈特默菲公司 | Compositions, methods and systems for inferring bovine breed |
| CN104611349A (en) * | 2014-11-24 | 2015-05-13 | 吉林大学 | FDFT1 gene key loci affecting Chinese simmental cattle fat deposition |
| CN104630341A (en) * | 2014-11-24 | 2015-05-20 | 吉林大学 | Chinese Simmental cattle FGF-1 gene as genetic markers of carcass meat quality |
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| CN101405404A (en) * | 2002-12-31 | 2009-04-08 | 迈特默菲公司 | Compositions, methods and systems for inferring bovine breed |
| CN104611349A (en) * | 2014-11-24 | 2015-05-13 | 吉林大学 | FDFT1 gene key loci affecting Chinese simmental cattle fat deposition |
| CN104630341A (en) * | 2014-11-24 | 2015-05-20 | 吉林大学 | Chinese Simmental cattle FGF-1 gene as genetic markers of carcass meat quality |
Non-Patent Citations (3)
| Title |
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| http://genome.ucsc.edu/cgi-bin/hgc?c=chr19&l=51333255&r=51333627&o=51333373&t=51333374&g=snp148&i=rs109238056&db=bosTau8;UCSC;《UCSC》;20160730;全文 * |
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